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Team:SUSTC-Shenzhen/Notebook/A-B Toxin/SDS PAGE
From 2014.igem.org
Notebook
Element of an endeavor
Contents |
A-B Toxin
2014 --to purify the proteinProtocol
Supplies and reagents
- Acrylamide solutions
- 12% resolving gel(because our protein separately are 43kDa and 69kDa, so we should use 12% resolving gel)
| components\volume | 5ml | 10ml | 15ml | 20ml | 25ml | 30ml | 40ml | 50ml |
|---|---|---|---|---|---|---|---|---|
| H2O | 1.6 | 3.3 | 4.9 | 6.6 | 8.2 | 9.9 | 31.2 | 16.5 |
| 30%acrylamide mix | 2.0 | 4.0 | 6.0 | 8.0 | 10.0 | 12.0 | 16.0 | 20.0 |
| Tris-Cl(1.5M,PH8.8) | 1.3 | 2.5 | 3.8 | 5.0 | 6.3 | 7.5 | 10.0 | 12.5 |
| SDS(10%) | 0.05 | 0.1 | 0.15 | 0.2 | 0.25 | 0.3 | 0.4 | 0.5 |
| 10%ammonium persulfate | 0.05 | 0.1 | 0.15 | 0.2 | 0.25 | 0.3 | 0.4 | 0.5 |
| TEMED | 0.002 | 0.004 | 0.006 | 0.008 | 0.01 | 0.012 | 0.016 | 0.02 |
- 5% stacking gel
| Components\Volume | 1ml | 2ml | 3ml | 4ml | 5ml | 6ml | 8ml | 10ml |
|---|---|---|---|---|---|---|---|---|
| H2O | 0.68 | 1.4 | 2.1 | 2.7 | 3.4 | 4.1 | 5.5 | 6.8 |
| 30%acrylamide mix | 0.17 | 0.33 | 0.5 | 0.67 | 0.83 | 1.0 | 1.3 | 1.7 |
| Tris-Cl(0.5M,PH6.8) | 0.13 | 0.25 | 0.38 | 0.5 | 0.63 | 0.75 | 1.0 | 1.25 |
| SDS(10%) | 0.01 | 0.02 | 0.03 | 0.04 | 0.05 | 0.06 | 0.08 | 0.1 |
| 10%ammonium persulfate | 0.01 | 0.02 | 0.03 | 0.04 | 0.05 | 0.06 | 0.08 | 0.1 |
| TEMED | 0.001 | 0.002 | 0.003 | 0.004 | 0.005 | 0.006 | 0.008 | 0.01 |
- Coomassie Brilliant Blue
- Coomassie Brilliant Blue R-250 1.25g, alcohol 250ml, acetic acid 80ml and ddH2O 670ml.
- destain buffer
- 40%alcohol+10%acetic acid
- ammonium persulfate in 10ml of H2O(10%w/v) and store at 4ºC
- 10%SDS
- ®Invitrogen Protein standard molecular-weight marker
- ®Invitrogen 5X SDS gel loading buffer
- TEMED(electrophoreis grade)
- Tris-HCl(0.5M,PH6.8),250ml
- Tris-HCl(1.5M,PH8.8),250ml
Pouring gel
- assembly the glass plate and fixation the equipment
- pour the resolving gel, and keep a suitable distance from the opening.
- cover a shell of ddH2O on the resolving gel until it solidify
- remove the water shell and add the stacking gel.
- add a Teflon comb into the stacking gel, when it become solid, remove it
Preparation of sample
prepare the samples in the appropriate volume of SDS gel-loading buffer and heat them into 100ºC for 3mins to denature the proteins
Running the SDS-PAGE gel
- add a suitable volume of denatured samples into the hole of gel.
- run the gel machine.
- when the process finished, remove the glass plate
- use the Coomassie Brilliant Blue to stain the gel
- use the destaining solution to wash for about 1hrs
- use the ddH2O to heat in 100ºC for 10mins.
