Aims
To test for expression of biomaterials in B.subtilis and to determine if the biomaterials when expressed are being secreted or are remaining within the cell. This can also be extended to find optimal conditions for production and secretion.
Equipment
Vertical Gel electrophoresis tank and equipment
Desktop centrifuge
Pipettes
Eppendorfs
Reagents
Protein Marker
LB medium (and antibiotics)
Protocol
Preparing a polyacrylamide gel
15% SDS-PAGE gels were prepared using protocols from the OWW,[http://openwetware.org/wiki/Keating:Experimental_Protocols:SDS-PAGE click this link]
Production of Samples
- B.subtilis transformed with biomaterials were used to inncolulate 5ml of LB media grown with suitable antibiotics.
- These samples were induced with 1mM IPTG at mid log phase (O.D.600 1.5) and grown overnight.
- In the following morning the 5ml samples were spun down at 5000g for 5 minutes. The media was collected and the removed from the pellet.
Loading and running the Gel
- Boil all samples at 95°C for 5 minutes
- Carefully mix each sample before loading by rubbing tube between fingers and load 15μL of sample into the wells, taking care to note which sample is in which well on which gel (if 2 are being loaded simultaneously).
- When loading to gels, load along in one direction, alternating between the two gels (if both used), but beginning with the rear gel.
- Be careful to load sample into a single well and not contaminate adjacent wells and avoid bubbles where possible
- Gel should be run for approximately 70 minutes at 130V, or until the blue gel front has been eluted from the bottom of the gel
- Check for bubble rising to ensure the gel is running correctly
Gel removal and Staining
- When the running buffer has been eluted from the gel or has reached the very bottom, turn off the current and machine
- Remove the lid of the PAGE tank and remove the gel holder from inside
- Release both gels from the holder and lay them down separately in their glass cases
- Carefully remove the glass cover and place well away from other glass objects
- Prepare a tray with a protein dye solution
- Slowly peel the gel off the glass plate and into the protein dye solution (using the adhesive ability of the water helps)
- Leave to stain for 20 minutes or overnight (if staining overnight, place a film or lid over the tray to reduce evaporation)
- To destain, the stain should be poured back into the stain bottle and the gel immersed in destain solution until the destain solution is bright blue
- At this point, pour off the old destain and add some fresh
- When the gel background is relatively colourless, the gel should be removed from destain and kept in water before imaging
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