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

  • 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

  • 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.

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