http://2008.igem.org/wiki/index.php?title=Team:Slovenia/Notebook/Methods&feed=atom&action=historyTeam:Slovenia/Notebook/Methods - Revision history2024-03-29T15:59:38ZRevision history for this page on the wikiMediaWiki 1.16.5http://2008.igem.org/wiki/index.php?title=Team:Slovenia/Notebook/Methods&diff=104938&oldid=prevSloGuy at 04:42, 30 October 20082008-10-30T04:42:46Z<p></p>
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</table>SloGuyhttp://2008.igem.org/wiki/index.php?title=Team:Slovenia/Notebook/Methods&diff=104811&oldid=prevAnze at 04:33, 30 October 20082008-10-30T04:33:23Z<p></p>
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</table>Anzehttp://2008.igem.org/wiki/index.php?title=Team:Slovenia/Notebook/Methods&diff=104498&oldid=prevAnze at 04:10, 30 October 20082008-10-30T04:10:31Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-size: 130%;"><span style="font-family: Arial;"><strong><u><span lang="EN-US">PROTEIN PRODUCTION, ISOLATION AND MODIFICATION</span></u></strong></span></span></span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-size: 130%;"><span style="font-family: Arial;"><strong><u><span lang="EN-US">PROTEIN PRODUCTION, ISOLATION AND MODIFICATION</span></u></strong></span></span></span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;">&nbsp;</span></span><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;">&nbsp;</span></span><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;"><span lang="EN-US">The complete coding regions of <i>H. pylori flaA</i> from the&nbsp;<i>H. pylori</i> SS1 strain and <i>fliC</i> from <i>Escherichia coli</i> K12 were amplified by PCR and cloned into AK3 or pET19b vector (Novagen). Chimeric flagellins were constructed via PCR ligation and Biobrick assembly methods. For the DNA cloning experiments <i>E.coli</i>&nbsp; DH<i>5</i>&alpha; strain was used. After DNA sequences verification <i>E.coli</i> BL21(DE3)pLysS were transformed with specific constructs for the overexpression of flagellar proteins.&nbsp;Cultures were grown in LB media (Luria-Bertani) supplemented with ampicillin&nbsp;at 37&deg;C with shaking at 180 rpm until the OD600 was 0.4-0.5.&nbsp;At this point temperature was shifted to 25&deg;C for maximal yield of native protein in supernatant. To induce protein expression 1 mM IPTG&nbsp;(isopropyl &beta;-D-1-thiogalactopyranoside) was added when OD600&nbsp; was 0.7-1. Bacterial cells were&nbsp;further cultivated overnight and then harvested by centrifugation (5000g,&nbsp;10 min at 4&deg;C). Bacteria were resuspended in lysis buffer (0.1% sodium deoxicholate, 10 mM Tris/HCl pH 8.0) with protease inhibitors (CPI, Sigma)&nbsp;and sonicated for&nbsp;homogenisation. After centrifugation, supernatant with soluble fraction of His-tagged recombinant flagellin was loaded on a pre-conditioned Ni-NTA column (Ni-NTA Agarose, Qiagen), which was then washed in 50 mM Tris/HCl, 100 mM NaCl, 20 mM imidazole, pH 8.0<del class="diffchange diffchange-inline">)</del>. Proteins were eluted with buffers, containing 50 mM Tris/HCl, 100 mM NaCl, and imidazole with concentrations of 50 mM and 250 mM. Fractions that&nbsp;spectrophotometrically indicated&nbsp;typical protein peaks at 280 nm were&nbsp;combined and concentrated. Purity and integrity of proteins was assessed by SDS-PAGE and&nbsp;by Western blot. Proteins were separated&nbsp;on 10% SDS-PAGE gels and stained with Coomassie Blue or transferred for&nbsp;60 min at 350 mA&nbsp;to nitrocellulose membranes (Amersham).&nbsp;Membranes were&nbsp;blocked with 0.2% I-Block (Tropix)&nbsp;in PBS-T (1x PBS/0,1% Tween-20) for 60 min, then incubated sequentially with primary mouse&nbsp;anti-His&nbsp;monoclonal antibody (Qiagen)&nbsp;for 90 min at a dilution of 1:1000 and peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology) for 45 min at a dilution of 1:3000 at room temperature. The signal was detected with chemiluminescence substrate (Pierce). As proteins were determined, we dialyzed them two times against 1x PBS buffer. Secondary structure was examined by CD spectroscopy.</span></span></span></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;"><span lang="EN-US">The complete coding regions of <i>H. pylori flaA</i> from the&nbsp;<i>H. pylori</i> SS1 strain and <i>fliC</i> from <i>Escherichia coli</i> K12 were amplified by PCR and cloned into AK3 or pET19b vector (Novagen). Chimeric flagellins were constructed via PCR ligation and Biobrick assembly methods. For the DNA cloning experiments <i>E. coli</i>&nbsp; DH<i>5</i>&alpha; strain was used. After DNA sequences verification <i>E. coli</i> BL21(DE3)pLysS were transformed with specific constructs for the overexpression of flagellar proteins.&nbsp;Cultures were grown in LB media (Luria-Bertani) supplemented with ampicillin&nbsp;at 37&deg;C with shaking at 180 rpm until the OD600 was 0.4-0.5.&nbsp;At this point temperature was shifted to 25&deg;C for maximal yield of native protein in supernatant. To induce protein expression 1 mM IPTG&nbsp;(isopropyl &beta;-D-1-thiogalactopyranoside) was added when OD600&nbsp; was 0.7-1. Bacterial cells were&nbsp;further cultivated overnight and then harvested by centrifugation (5000g,&nbsp;10 min at 4&deg;C). Bacteria were resuspended in lysis buffer (0.1% sodium deoxicholate, 10 mM Tris/HCl pH 8.0) with protease inhibitors (CPI, Sigma)&nbsp;and sonicated for&nbsp;homogenisation. After centrifugation, supernatant with soluble fraction of His-tagged recombinant flagellin was loaded on a pre-conditioned Ni-NTA column (Ni-NTA Agarose, Qiagen), which was then washed in 50 mM Tris/HCl, 100 mM NaCl, 20 mM imidazole, pH 8.0. Proteins were eluted with buffers, containing 50 mM Tris/HCl, 100 mM NaCl, and imidazole with concentrations of 50 mM and 250 mM. Fractions that&nbsp;spectrophotometrically indicated&nbsp;typical protein peaks at 280 nm were&nbsp;combined and concentrated. Purity and integrity of proteins was assessed by SDS-PAGE and&nbsp;by Western blot. Proteins were separated&nbsp;on 10% SDS-PAGE gels and stained with Coomassie Blue or transferred for&nbsp;60 min at 350 mA&nbsp;to nitrocellulose membranes (Amersham).&nbsp;Membranes were&nbsp;blocked with 0.2% I-Block (Tropix)&nbsp;in PBS-T (1x PBS/0,1% Tween-20) for 60 min, then incubated sequentially with primary mouse&nbsp;anti-His&nbsp;monoclonal antibody (Qiagen)&nbsp;for 90 min at a dilution of 1:1000 and peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology) for 45 min at a dilution of 1:3000 at room temperature. The signal was detected with chemiluminescence substrate (Pierce). As proteins were determined, we dialyzed them two times against 1x PBS buffer. Secondary structure was examined by CD spectroscopy.</span></span></span></p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;"><strong><span lang="EN-US">CIRCULAR DICHROISM</span></strong></span></span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;"><strong><span lang="EN-US">CIRCULAR DICHROISM</span></strong></span></span></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span lang="EN-US">Circular dichroism (CD) is a form of spectroscopy based on the differential absorption of left- and right-handed circularly polarized light. It can be used to help determine the structure of macromolecules (including the secondary structure of proteins).&nbsp;This method was used to confirm that intact protein with it's proper secondary structure was isolated. CD <del class="diffchange diffchange-inline">pectra </del>were&nbsp;&nbsp;taken the far-UV region between 190 and&nbsp;250 nm on a Chirascan CD spectrometer (Applied Photophysics). The cell path length used was&nbsp;1 mm&nbsp;with sample concentration 0.5 mg/ml in MQ water.</span></span></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span lang="EN-US">Circular dichroism (CD) is a form of spectroscopy based on the differential absorption of left- and right-handed circularly polarized light. It can be used to help determine the structure of macromolecules (including the secondary structure of proteins).&nbsp;This method was used to confirm that intact protein with it's proper secondary structure was isolated. CD <ins class="diffchange diffchange-inline">spectra </ins>were&nbsp;&nbsp;taken the far-UV region between 190 and&nbsp;250 nm on a Chirascan CD spectrometer (Applied Photophysics). The cell path length used was&nbsp;1 mm&nbsp;with sample concentration 0.5 mg/ml in MQ water.</span></span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;"><strong><span lang="EN-US">FLUORESCENT PROTEIN LABELING</span></strong></span></span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;"><strong><span lang="EN-US">FLUORESCENT PROTEIN LABELING</span></strong></span></span></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;"><span lang="EN-US">Proteins were fluorescently&nbsp;<del class="diffchange diffchange-inline">labelled </del>with Alexa Fluor 555 hydrazide (Molecular Probes),&nbsp;incubated with&nbsp;100 mM natrium &nbsp;borate &nbsp;(pH 8.5) for 2 h at room temperature with shaking (protected from light). Afterwards, 10-fold excess of Tris buffer (pH=7) was added to deactivate remainder of Alexa dye, after it&nbsp; has reacted with primary amines.&nbsp; Finally, to remove unbound Alexa Fluor 555,&nbsp; fluorescently labeled proteins were extensively dialyzed against PBS buffer.&nbsp;</span></span></span></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;"><span lang="EN-US">Proteins were fluorescently&nbsp;<ins class="diffchange diffchange-inline">labeled </ins>with Alexa Fluor 555 hydrazide (Molecular Probes),&nbsp;incubated with&nbsp;100 mM natrium &nbsp;borate &nbsp;(pH 8.5) for 2 h at room temperature with shaking (protected from light). Afterwards, 10-fold excess of Tris buffer (pH=7) was added to deactivate remainder of Alexa dye, after it&nbsp; has reacted with primary amines.&nbsp; Finally, to remove unbound Alexa Fluor 555,&nbsp; fluorescently labeled proteins were extensively dialyzed against PBS buffer.&nbsp;</span></span></span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-size: 130%;"><span style="font-family: Arial;"><strong><span lang="EN-US"><u>ELISA</u></span></strong></span></span></span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-size: 130%;"><span style="font-family: Arial;"><strong><span lang="EN-US"><u>ELISA</u></span></strong></span></span></span></p></div></td></tr>
</table>Anzehttp://2008.igem.org/wiki/index.php?title=Team:Slovenia/Notebook/Methods&diff=101256&oldid=prevMonika at 02:12, 30 October 20082008-10-30T02:12:41Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><p><span style="color: rgb(0, 0, 0);"><span style="font-size: 130%;">&nbsp;</span></span></p></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">For detecting production of cytokines upon cell activation with&nbsp;DNA TLR vaccines&nbsp;we used real-time PCR and an Multiplex Fluorescent Bead Immunoassay&nbsp;(Bender MedSystems)&nbsp;for quantitative detection of human cytokines by flow cytometry.</span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">For detecting production of cytokines upon cell activation with&nbsp;DNA TLR vaccines&nbsp;we used real-time PCR and an Multiplex Fluorescent Bead Immunoassay&nbsp;(Bender MedSystems)&nbsp;for quantitative detection of human cytokines by flow cytometry.</span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);">HEK293 cells were seeded into a 12-well plate and transfected with the indicated DNA TLR vaccines&nbsp;or wtTLR plasmid controls. 24 h post transfection the DNA TLR vaccine transfected cells were lysed for RNA extraction and positive control (wtTLR-vector transfected) cells were stimulated for 4 h with the corresponding ligands (LPS and poly(I:C)) and then lysed for RNA extraction. Supernatants from the same cells were collected for the Flurescent Bead Immunoassay.</span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);">HEK293 cells were seeded into a 12-well plate and transfected with the indicated DNA TLR vaccines&nbsp;or wtTLR plasmid controls. 24 h post transfection the DNA TLR vaccine transfected cells were lysed for RNA extraction and positive control (wtTLR-vector transfected) cells were stimulated for 4 h with the corresponding ligands (LPS and poly(I:C)) and then lysed for RNA extraction. Supernatants from the same cells were collected for the Flurescent Bead Immunoassay.</span></p></div></td></tr>
</table>Monikahttp://2008.igem.org/wiki/index.php?title=Team:Slovenia/Notebook/Methods&diff=101099&oldid=prevMonika at 02:07, 30 October 20082008-10-30T02:07:04Z<p></p>
<a href="http://2008.igem.org/wiki/index.php?title=Team:Slovenia/Notebook/Methods&diff=101099&oldid=100829">Show changes</a>Monikahttp://2008.igem.org/wiki/index.php?title=Team:Slovenia/Notebook/Methods&diff=100829&oldid=prevMonika at 01:57, 30 October 20082008-10-30T01:57:44Z<p></p>
<a href="http://2008.igem.org/wiki/index.php?title=Team:Slovenia/Notebook/Methods&diff=100829&oldid=100709">Show changes</a>Monikahttp://2008.igem.org/wiki/index.php?title=Team:Slovenia/Notebook/Methods&diff=100709&oldid=prevMonika at 01:52, 30 October 20082008-10-30T01:52:46Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;"><strong><span lang="EN-US">CIRCULAR DICHROISM</span></strong></span></span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;"><strong><span lang="EN-US">CIRCULAR DICHROISM</span></strong></span></span></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span lang="EN-US">Circular dichroism (CD) is a form of spectroscopy based on the differential absorption of left- and right-handed circularly polarized light. It can be used to help determine the structure of macromolecules (including the secondary structure of proteins).&nbsp;This method was used to confirm that intact protein with it's proper secondary structure was isolated. CD pectra were&nbsp;&nbsp;taken the far-UV region between 190 and&nbsp;250 nm on a Chirascan CD spectrometer (Applied Photophysics). The cell path length used was&nbsp;1 mm&nbsp;with sample concentration 0.5 mg/ml in MQ water.</span></span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span lang="EN-US">Circular dichroism (CD) is a form of spectroscopy based on the differential absorption of left- and right-handed circularly polarized light. It can be used to help determine the structure of macromolecules (including the secondary structure of proteins).&nbsp;This method was used to confirm that intact protein with it's proper secondary structure was isolated. CD pectra were&nbsp;&nbsp;taken the far-UV region between 190 and&nbsp;250 nm on a Chirascan CD spectrometer (Applied Photophysics). The cell path length used was&nbsp;1 mm&nbsp;with sample concentration 0.5 mg/ml in MQ water.</span></span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;"><strong><span lang="EN-US">FLUORESCENT PROTEIN LABELING</span></strong></span></span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;"><strong><span lang="EN-US">FLUORESCENT PROTEIN LABELING</span></strong></span></span></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;"><span lang="EN-US">Proteins were fluorescently&nbsp;labelled with Alexa Fluor 555 hydrazide (Molecular Probes),&nbsp;incubated with&nbsp;100 mM natrium &nbsp;borate &nbsp;(pH 8.5) for 2 h at room temperature with shaking (protected from light). Afterwards, 10-fold excess of Tris buffer (pH=7) was added to deactivate remainder of Alexa dye, after it&nbsp; has reacted with primary amines.&nbsp; Finally, to remove unbound Alexa Fluor 555,&nbsp; fluorescently labeled proteins were extensively dialyzed against PBS buffer.&nbsp;</span></span></span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;"><span lang="EN-US">Proteins were fluorescently&nbsp;labelled with Alexa Fluor 555 hydrazide (Molecular Probes),&nbsp;incubated with&nbsp;100 mM natrium &nbsp;borate &nbsp;(pH 8.5) for 2 h at room temperature with shaking (protected from light). Afterwards, 10-fold excess of Tris buffer (pH=7) was added to deactivate remainder of Alexa dye, after it&nbsp; has reacted with primary amines.&nbsp; Finally, to remove unbound Alexa Fluor 555,&nbsp; fluorescently labeled proteins were extensively dialyzed against PBS buffer.&nbsp;</span></span></span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-size: 130%;"><span style="font-family: Arial;"><strong><span lang="EN-US"><u>ELISA</u></span></strong></span></span></span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-size: 130%;"><span style="font-family: Arial;"><strong><span lang="EN-US"><u>ELISA</u></span></strong></span></span></span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);"><span style="font-size: 100%;">ELISA (Enzyme-Linked ImmunoSorbent Assay) is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. In our experiments ELISA was used to determine mouse serum IgG antibody response to the recombinant protein&nbsp;vaccination. 96-well microtiter plates were coated either with 50&nbsp;&micro;l&nbsp;of each recombinant protein antigen&nbsp;(conc. 10 &micro;g/ml) or heat killed <em>H. pylori</em>&nbsp;preparation from 50 million cells in the volume of 50 &micro;l&nbsp;overnight at 4&deg;C. After blocking&nbsp;&nbsp;with 3%&nbsp;BSA in PBST (150 mM NaCl, 7.5 mM Na<sub>2</sub>HPO<sub>4</sub>, 2.5 mM NaH<sub>2</sub>PO<sub>4</sub>, 0.05% Tween-20) overnight in a humid atmosphere at 4&deg;C and washing three times with PBST, appropriate dilutions of sera were&nbsp;added&nbsp;to the wells, and incubated for 90 min at 37&deg;C to&nbsp;enable&nbsp;specific IgG antibody&nbsp;binding to immobilized antigens. Following a washing step,&nbsp;horse radish peroxidase-conjugated secondary antibodies with a specifity for the mouse IgG class of antibodies&nbsp;were added at a dilution of&nbsp;1:3000. After another 90 min incubation at 37&deg;C&nbsp;plates were washed again to remove unbound secondary antibodies. Finally, ABTS (Sigma) substrate was added&nbsp;and&nbsp;after 20 min incubation the&nbsp;reaction was stopped with 1% SDS.&nbsp;Absorbance values were&nbsp;quantified at&nbsp;450 nm in a multilabel plate reader Mithras (Berthold Technologies).</span></span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);"><span style="font-size: 100%;">ELISA (Enzyme-Linked ImmunoSorbent Assay) is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. In our experiments ELISA was used to determine mouse serum IgG antibody response to the recombinant protein&nbsp;vaccination. 96-well microtiter plates were coated either with 50&nbsp;&micro;l&nbsp;of each recombinant protein antigen&nbsp;(conc. 10 &micro;g/ml) or heat killed <em>H. pylori</em>&nbsp;preparation from 50 million cells in the volume of 50 &micro;l&nbsp;overnight at 4&deg;C. After blocking&nbsp;&nbsp;with 3%&nbsp;BSA in PBST (150 mM NaCl, 7.5 mM Na<sub>2</sub>HPO<sub>4</sub>, 2.5 mM NaH<sub>2</sub>PO<sub>4</sub>, 0.05% Tween-20) overnight in a humid atmosphere at 4&deg;C and washing three times with PBST, appropriate dilutions of sera were&nbsp;added&nbsp;to the wells, and incubated for 90 min at 37&deg;C to&nbsp;enable&nbsp;specific IgG antibody&nbsp;binding to immobilized antigens. Following a washing step,&nbsp;horse radish peroxidase-conjugated secondary antibodies with a specifity for the mouse IgG class of antibodies&nbsp;were added at a dilution of&nbsp;1:3000. After another 90 min incubation at 37&deg;C&nbsp;plates were washed again to remove unbound secondary antibodies. Finally, ABTS (Sigma) substrate was added&nbsp;and&nbsp;after 20 min incubation the&nbsp;reaction was stopped with 1% SDS.&nbsp;Absorbance values were&nbsp;quantified at&nbsp;450 nm in a multilabel plate reader Mithras (Berthold Technologies).</span></span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-size: 130%;"><strong><u><span lang="EN-US">FLOW CYTOMETRY</span></u></strong></span></span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-size: 130%;"><strong><u><span lang="EN-US">FLOW CYTOMETRY</span></u></strong></span></span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span lang="EN-US">Flow cytometry employs instrumentation measuring the fluorescence on individual cells. Cells are suspended in individual droplets, squirted past a laser that excites any fluorescent dye in them and past a detector that measures the fluorescence. Fluorescence measurement was carried out on a Epics Altra (Beckman-Coulter Electronics) Flow Cytometer Cell Sorter.</span></span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span lang="EN-US">Flow cytometry employs instrumentation measuring the fluorescence on individual cells. Cells are suspended in individual droplets, squirted past a laser that excites any fluorescent dye in them and past a detector that measures the fluorescence. Fluorescence measurement was carried out on a Epics Altra (Beckman-Coulter Electronics) Flow Cytometer Cell Sorter.</span></span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><strong>REACTIVITY OF SERUM ANTIBODIES WITH</strong> <em><strong>H. pylori</strong></em></span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><strong>REACTIVITY OF SERUM ANTIBODIES WITH</strong> <em><strong>H. pylori</strong></em></span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span lang="EN-US">In our experiment we used fluorescently labeled antibody</span> fragment goat F(ab)2 anti-mouse IgG-PE to detect IgG reaction with <em>H. pylori</em>. Collected <i>H. pylori</i> cells were washed with FACS (10x PBS, 0.5% BSA, 0.05% Azide)&nbsp;buffer and&nbsp;incubated &nbsp;for 30 min on ice &nbsp;in 100&nbsp;&micro;l FACS buffer containing&nbsp;&nbsp;8 &micro;l serum. After that bacteria were&nbsp;washed again and incubated for 30 min on ice in FACS buffer containing&nbsp;F(ab)2 goat anti-mouse IgG fragment, conjugated to phycoerythrin (Beckman Coulter), to a final concentration 0.01 mg/ml. After another washing and resuspension step, samples were placed in&nbsp;reaction tubes and analyzed with flow cytometry.&nbsp;</span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span lang="EN-US">In our experiment we used fluorescently labeled antibody</span> fragment goat F(ab)2 anti-mouse IgG-PE to detect IgG reaction with <em>H. pylori</em>. Collected <i>H. pylori</i> cells were washed with FACS (10x PBS, 0.5% BSA, 0.05% Azide)&nbsp;buffer and&nbsp;incubated &nbsp;for 30 min on ice &nbsp;in 100&nbsp;&micro;l FACS buffer containing&nbsp;&nbsp;8 &micro;l serum. After that bacteria were&nbsp;washed again and incubated for 30 min on ice in FACS buffer containing&nbsp;F(ab)2 goat anti-mouse IgG fragment, conjugated to phycoerythrin (Beckman Coulter), to a final concentration 0.01 mg/ml. After another washing and resuspension step, samples were placed in&nbsp;reaction tubes and analyzed with flow cytometry.&nbsp;</span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><b>FLOW CYTOMETRY PROTOCOL FOR INTRACELLULAR STAINING OF HEK293T</b></span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><b>FLOW CYTOMETRY PROTOCOL FOR INTRACELLULAR STAINING OF HEK293T</b></span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>&nbsp;</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>&nbsp;</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><b>FLOW CYTOMETRY PROTOCOL FOR SURFACE STAINING OF HEK293T&nbsp;</b></span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><b>FLOW CYTOMETRY PROTOCOL FOR SURFACE STAINING OF HEK293T&nbsp;</b></span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);">48h prior intracellular staining HEK293T cells were transfected with 2000 ng of indicated construct using GeneJuice Transfection Reagent (Novagen). We began surface staining with resuspension of cells in PBS + 3% FBS. Then we fixed the cells 10 min at room temperature by adding 4% paraformaldehyde. Cells were washed two-times with PBS + 3% FBS. Cells were then stained with primary antibodies (mouse anti-HA antibodies, Invivogen, final concentration 0.08 mg/ml) 40 min at 4&deg;C and then with secondary antibodies (goat anti-mouse IgG-PE, Beck.Coul., final concentration 0.01 mg/ml) for 30 min at 4&deg;C. After that we washed cells two-times and analyzed them on flow cytometer.</span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);">48h prior intracellular staining HEK293T cells were transfected with 2000 ng of indicated construct using GeneJuice Transfection Reagent (Novagen). We began surface staining with resuspension of cells in PBS + 3% FBS. Then we fixed the cells 10 min at room temperature by adding 4% paraformaldehyde. Cells were washed two-times with PBS + 3% FBS. Cells were then stained with primary antibodies (mouse anti-HA antibodies, Invivogen, final concentration 0.08 mg/ml) 40 min at 4&deg;C and then with secondary antibodies (goat anti-mouse IgG-PE, Beck.Coul., final concentration 0.01 mg/ml) for 30 min at 4&deg;C. After that we washed cells two-times and analyzed them on flow cytometer.</span></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></del></div></td><td colspan="2"> </td></tr>
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</table>Monikahttp://2008.igem.org/wiki/index.php?title=Team:Slovenia/Notebook/Methods&diff=100637&oldid=prevRjerala at 01:50, 30 October 20082008-10-30T01:50:28Z<p></p>
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</table>Rjeralahttp://2008.igem.org/wiki/index.php?title=Team:Slovenia/Notebook/Methods&diff=100615&oldid=prevMonika at 01:49, 30 October 20082008-10-30T01:49:48Z<p></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 01:49, 30 October 2008</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-size: 130%;"><span style="font-family: Arial;"><strong><u><span lang="EN-US">PROTEIN PRODUCTION, ISOLATION AND MODIFICATION</span></u></strong></span></span></span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-size: 130%;"><span style="font-family: Arial;"><strong><u><span lang="EN-US">PROTEIN PRODUCTION, ISOLATION AND MODIFICATION</span></u></strong></span></span></span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;">&nbsp;</span></span><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;">&nbsp;</span></span><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;"><span lang="EN-US">The complete coding regions of <i>H. pylori flaA</i> from the&nbsp;<i>H. pylori</i> SS1 strain and <i>fliC</i> from <i>Escherichia coli</i> K12 were amplified by PCR and cloned into AK3 or pET19b vector (Novagen). Chimeric flagellins were constructed via PCR ligation and Biobrick assembly methods. For the DNA cloning experiments <i>E.coli</i>&nbsp; DH<i>5</i>&alpha; strain was used. After DNA sequences verification <i>E.coli</i> BL21(DE3)pLysS were transformed with specific constructs for the overexpression of flagellar proteins.&nbsp;Cultures were grown in LB media (Luria-Bertani) supplemented with ampicillin&nbsp;at 37&deg;C with shaking at 180 rpm until the OD600 was 0.4-0.5.&nbsp;At this point temperature was shifted to 25&deg;C for maximal yield of native protein in supernatant. To induce protein expression 1 mM IPTG&nbsp;(isopropyl &beta;-D-1-thiogalactopyranoside) was added when OD600&nbsp; was 0.7-1. Bacterial cells were&nbsp;further cultivated overnight and then harvested by centrifugation (5000g,&nbsp;10 min at 4&deg;C). Bacteria were resuspended in lysis buffer (0.1% sodium deoxicholate, 10 mM Tris/HCl pH 8.0) with protease inhibitors (CPI, Sigma)&nbsp;and sonicated for&nbsp;homogenisation. After centrifugation, supernatant with soluble fraction of His-tagged recombinant flagellin was loaded on a pre-conditioned Ni-NTA column (Ni-NTA Agarose, Qiagen), which was then washed in 50 mM Tris/HCl, 100 mM NaCl, 20 mM imidazole, pH 8.0). Proteins were eluted with buffers, containing 50 mM Tris/HCl, 100 mM NaCl, and imidazole with concentrations of 50 mM and 250 mM. Fractions that&nbsp;spectrophotometrically indicated&nbsp;typical protein peaks at 280 nm were&nbsp;combined and concentrated. Purity and integrity of proteins was assessed by SDS-PAGE and&nbsp;by Western blot. Proteins were separated&nbsp;on 10% SDS-PAGE gels and stained with Coomassie Blue or transferred for&nbsp;60 min at 350 mA&nbsp;to nitrocellulose membranes (Amersham).&nbsp;Membranes were&nbsp;blocked with 0.2% I-Block (Tropix)&nbsp;in PBS-T (1x PBS/0,1% Tween-20) for 60 min, then incubated sequentially with primary mouse&nbsp;anti-His&nbsp;monoclonal antibody (Qiagen)&nbsp;for 90 min at a dilution of 1:1000 and peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology) for 45 min at a dilution of 1:3000 at room temperature. The signal was detected with chemiluminescence substrate (Pierce). As proteins were determined, we dialyzed them two times against 1x PBS buffer. Secondary structure was examined by CD spectroscopy.</span></span></span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;"><span lang="EN-US">The complete coding regions of <i>H. pylori flaA</i> from the&nbsp;<i>H. pylori</i> SS1 strain and <i>fliC</i> from <i>Escherichia coli</i> K12 were amplified by PCR and cloned into AK3 or pET19b vector (Novagen). Chimeric flagellins were constructed via PCR ligation and Biobrick assembly methods. For the DNA cloning experiments <i>E.coli</i>&nbsp; DH<i>5</i>&alpha; strain was used. After DNA sequences verification <i>E.coli</i> BL21(DE3)pLysS were transformed with specific constructs for the overexpression of flagellar proteins.&nbsp;Cultures were grown in LB media (Luria-Bertani) supplemented with ampicillin&nbsp;at 37&deg;C with shaking at 180 rpm until the OD600 was 0.4-0.5.&nbsp;At this point temperature was shifted to 25&deg;C for maximal yield of native protein in supernatant. To induce protein expression 1 mM IPTG&nbsp;(isopropyl &beta;-D-1-thiogalactopyranoside) was added when OD600&nbsp; was 0.7-1. Bacterial cells were&nbsp;further cultivated overnight and then harvested by centrifugation (5000g,&nbsp;10 min at 4&deg;C). Bacteria were resuspended in lysis buffer (0.1% sodium deoxicholate, 10 mM Tris/HCl pH 8.0) with protease inhibitors (CPI, Sigma)&nbsp;and sonicated for&nbsp;homogenisation. After centrifugation, supernatant with soluble fraction of His-tagged recombinant flagellin was loaded on a pre-conditioned Ni-NTA column (Ni-NTA Agarose, Qiagen), which was then washed in 50 mM Tris/HCl, 100 mM NaCl, 20 mM imidazole, pH 8.0). Proteins were eluted with buffers, containing 50 mM Tris/HCl, 100 mM NaCl, and imidazole with concentrations of 50 mM and 250 mM. Fractions that&nbsp;spectrophotometrically indicated&nbsp;typical protein peaks at 280 nm were&nbsp;combined and concentrated. Purity and integrity of proteins was assessed by SDS-PAGE and&nbsp;by Western blot. Proteins were separated&nbsp;on 10% SDS-PAGE gels and stained with Coomassie Blue or transferred for&nbsp;60 min at 350 mA&nbsp;to nitrocellulose membranes (Amersham).&nbsp;Membranes were&nbsp;blocked with 0.2% I-Block (Tropix)&nbsp;in PBS-T (1x PBS/0,1% Tween-20) for 60 min, then incubated sequentially with primary mouse&nbsp;anti-His&nbsp;monoclonal antibody (Qiagen)&nbsp;for 90 min at a dilution of 1:1000 and peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology) for 45 min at a dilution of 1:3000 at room temperature. The signal was detected with chemiluminescence substrate (Pierce). As proteins were determined, we dialyzed them two times against 1x PBS buffer. Secondary structure was examined by CD spectroscopy.</span></span></span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;"><strong><span lang="EN-US">CIRCULAR DICHROISM</span></strong></span></span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-family: Arial;"><strong><span lang="EN-US">CIRCULAR DICHROISM</span></strong></span></span></p></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-size: 130%;"><span style="font-family: Arial;"><strong><span lang="EN-US">ELISA</span></strong></span></span></span></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-size: 130%;"><span style="font-family: Arial;"><strong><span lang="EN-US"<ins class="diffchange diffchange-inline">><u</ins>>ELISA<ins class="diffchange diffchange-inline"></u></ins></span></strong></span></span></span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);">&nbsp;</span></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);"><span style="font-size: 100%;">ELISA (Enzyme-Linked ImmunoSorbent Assay) is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. In our experiments ELISA was used to determine mouse serum IgG antibody response to the recombinant protein&nbsp;vaccination. 96-well microtiter plates were coated either with 50&nbsp;&micro;l&nbsp;of each recombinant protein antigen&nbsp;(conc. 10 &micro;g/ml) or heat killed <em>H. pylori</em>&nbsp;preparation from 50 million cells in the volume of 50 &micro;l&nbsp;overnight at 4&deg;C. After blocking&nbsp;&nbsp;with 3%&nbsp;BSA in PBST (150 mM NaCl, 7.5 mM Na<sub>2</sub>HPO<sub>4</sub>, 2.5 mM NaH<sub>2</sub>PO<sub>4</sub>, 0.05% Tween-20) overnight in a humid atmosphere at 4&deg;C and washing three times with PBST, appropriate dilutions of sera were&nbsp;added&nbsp;to the wells, and incubated for 90 min at 37&deg;C to&nbsp;enable&nbsp;specific IgG antibody&nbsp;binding to immobilized antigens. Following a washing step,&nbsp;horse radish peroxidase-conjugated secondary antibodies with a specifity for the mouse IgG class of antibodies&nbsp;were added at a dilution of&nbsp;1:3000. After another 90 min incubation at 37&deg;C&nbsp;plates were washed again to remove unbound secondary antibodies. Finally, ABTS (Sigma) substrate was added&nbsp;and&nbsp;after 20 min incubation the&nbsp;reaction was stopped with 1% SDS.&nbsp;Absorbance values were&nbsp;quantified at&nbsp;450 nm in a multilabel plate reader Mithras (Berthold Technologies).</span></span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><span style="color: rgb(0, 0, 0);"><span style="font-size: 100%;">ELISA (Enzyme-Linked ImmunoSorbent Assay) is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. In our experiments ELISA was used to determine mouse serum IgG antibody response to the recombinant protein&nbsp;vaccination. 96-well microtiter plates were coated either with 50&nbsp;&micro;l&nbsp;of each recombinant protein antigen&nbsp;(conc. 10 &micro;g/ml) or heat killed <em>H. pylori</em>&nbsp;preparation from 50 million cells in the volume of 50 &micro;l&nbsp;overnight at 4&deg;C. After blocking&nbsp;&nbsp;with 3%&nbsp;BSA in PBST (150 mM NaCl, 7.5 mM Na<sub>2</sub>HPO<sub>4</sub>, 2.5 mM NaH<sub>2</sub>PO<sub>4</sub>, 0.05% Tween-20) overnight in a humid atmosphere at 4&deg;C and washing three times with PBST, appropriate dilutions of sera were&nbsp;added&nbsp;to the wells, and incubated for 90 min at 37&deg;C to&nbsp;enable&nbsp;specific IgG antibody&nbsp;binding to immobilized antigens. Following a washing step,&nbsp;horse radish peroxidase-conjugated secondary antibodies with a specifity for the mouse IgG class of antibodies&nbsp;were added at a dilution of&nbsp;1:3000. After another 90 min incubation at 37&deg;C&nbsp;plates were washed again to remove unbound secondary antibodies. Finally, ABTS (Sigma) substrate was added&nbsp;and&nbsp;after 20 min incubation the&nbsp;reaction was stopped with 1% SDS.&nbsp;Absorbance values were&nbsp;quantified at&nbsp;450 nm in a multilabel plate reader Mithras (Berthold Technologies).</span></span></p></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-size: 130%;"><strong>CYTOKINE TESTING</strong></span></span></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span style="font-size: 130%;"><strong<ins class="diffchange diffchange-inline">><u</ins>>CYTOKINE TESTING<ins class="diffchange diffchange-inline"></u></ins></strong></span></span></p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;">&nbsp;</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span lang="EN-US">Each mouse was placed in a special ventilated and heated cage to enable peripheral vasodilatation for faster and easier blood sampling. The animal was then fixed in a restrainer and local anesthetic ethyl chloride was applied to the end of the tail. About 80-100 ml of blood was collected from the tail end and the tail tip was sealed with silver nitrate to stop bleeding.</span></span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="MsoNormal" style="margin: 0cm 0cm 0pt; text-align: justify;"><span style="color: rgb(0, 0, 0);"><span lang="EN-US">Each mouse was placed in a special ventilated and heated cage to enable peripheral vasodilatation for faster and easier blood sampling. The animal was then fixed in a restrainer and local anesthetic ethyl chloride was applied to the end of the tail. About 80-100 ml of blood was collected from the tail end and the tail tip was sealed with silver nitrate to stop bleeding.</span></span></p></div></td></tr>
</table>Monikahttp://2008.igem.org/wiki/index.php?title=Team:Slovenia/Notebook/Methods&diff=99749&oldid=prevRjerala at 01:11, 30 October 20082008-10-30T01:11:41Z<p></p>
<a href="http://2008.igem.org/wiki/index.php?title=Team:Slovenia/Notebook/Methods&diff=99749&oldid=97676">Show changes</a>Rjerala