Team:SUSTC-Shenzhen/Notebook/A-B Toxin/SDS PAGE

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Team SUSTC-Shenzhen

Notebook

Element of an endeavor

Contents


A-B Toxin

2014 --to purify the protein

protocol

supplies and reagents

  1. Acrylamide solutions
    • 12% resolving gel(because our protein separately are 43kDa and 69kDa, so we should use 12% resolving gel)

components\volume5ml10ml15ml20ml25ml30ml40ml50ml
H2O1.63.34.96.68.29.931.216.5
30%acrylamide mix2.04.06.08.010.012.016.020.0
Tris-Cl(1.5M,PH8.8)1.32.53.85.06.37.510.012.5
SDS(10%)0.050.10.150.20.250.30.40.5
10%ammonium persulfate0.050.10.150.20.250.30.40.5
TEMED0.0020.0040.0060.0080.010.0120.0160.02

    • 5% stacking gel

Components\Volume1ml2ml3ml4ml5ml6ml8ml10ml
H2O0.681.42.12.73.44.15.56.8
30%acrylamide mix0.170.330.50.670.831.01.31.7
Tris-Cl(0.5M,PH6.8)0.130.250.380.50.630.751.01.25
SDS(10%)0.010.020.030.040.050.060.080.1
10%ammonium persulfate0.010.020.030.040.050.060.080.1
TEMED0.0010.0020.0030.0040.0050.0060.0080.01

  1. Coomassie Brilliant Blue
    • Coomassie Brilliant Blue R-250 1.25g, alcohol 250ml, acetic acid 80ml and ddH2O 670ml.
  2. destain buffer
    • 40%alcohol+10%acetic acid
  3. ammonium persulfate in 10ml of H2O(10%w/v) and store at 4ºC
  4. 10%SDS
  5. ®Invitrogen Protein standard molecular-weight marker
  6. ®Invitrogen 5X SDS gel loading buffer
  7. TEMED(electrophoreis grade)
  8. Tris-HCl(0.5M,PH6.8),250ml
  9. Tris-HCl(1.5M,PH8.8),250ml

pouring gel

  1. assembly the glass plate and fixation the equipment
  2. pour the resolving gel, and keep a suitable distance from the opening.
  3. cover a shell of ddH2O on the resolving gel until it solidify
  4. remove the water shell and add the stacking gel.
  5. 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

  1. add a suitable volume of denatured samples into the hole of gel.
  2. run the gel machine.
  3. when the process finished, remove the glass plate
  4. use the Coomassie Brilliant Blue to stain the gel
  5. use the destaining solution to wash for about 1hrs
  6. use the ddH2O to heat in 100ºC for 10mins.

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