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RESEARCH DIVISION Laboratory Manual

 


 


SDS-PAGE GelsPreparation of SDS-PAGE Gels.

  1. Clean glass plates with ethanol and assemble casting stand, see Biorad instruction manual
  2. Mix solutions for lower gel in order shown ie. TEMED last and pour into plates leaving about 2cm at the top. There should be sufficient room to allow such that when the comb is inserted later there is about 5-8mm between the floor of the well and the top of the lower gel.
  3. Carefully overlay with DDW and allow to set. Once the gel has set it can be wrapped in gladwrap and stored for several days at least in the fridge.
  4. To continue remove overlay water, mix solutions for upper gel in order shown and overlay lower gel. Insert comb and allow to set for 15-20'. Generally the upper gel should not be prepared until the samples are ready as there is a pH difference between the two gels which will diffuse with time.
  5. Assemble running unit, see Biorad instruction manual and aspirate bubbles and liquid from wells. Do this immediately before samples are ready as the wells will dry out quite quickly.
  6. Load 10Ál - 25Ál (10Ál for a 15 tooth mini comb) and then overlay samples with 1 x SDS running buffer. Empty wells should have 10-15Ál sample buffer added and be overlayed as with the test samples so the gel runs straight. Flood the upper chamber by carefully adding 1 x  SDS running buffer to unit. Avoid pouring the buffer directly onto the wells.
  7. Run gels 200V for 30-45 min until dye front is at the bottom of gel. (Large BioRad gels -15 x 15cm - are run at 35mA per gel).
  8. See below for Western transfers. For gels to be stained, stain for 20 min with Coomassie blue solution (see below), then distain in strong distain (50% methanol, 10% acetic acid) 20 min and leave in 10% acetic acid.
Reagents for SDS PAGE
  1. 30% acryl/bis-acrylamide Bis: acrylamide 1:36
  513.5 ml 342.3ml
acrylamide 150 g 100g
bis-acrylamide 4.1 g 2.73g
DDW to 513.5 ml 342.3ml
- Filter and store at 4░
- Use Biorad acrylamide, bis-acrylamide
- Alternatively: Commercial pre-made acrylamide solutions can be used. These are usually supplied as a 40% stock and should be diluted to 30% for the subsequent protocols.
- The bis-acrylamide:acrylamide ratio should be 1:37.5 in the pre-made solution.
  1. SDS sample buffer 1x (clear) pH 6.8 250 ml total
Trizma base 1.9 g
Glycerol 31.42g
SDS 5.75 g
DDW to 250 ml
  1. 2X SDS SB:
- As above and then to final volume of 125ml
- Adjust pH with concentrated HCl.
- NOTE. Add the HCl slowly once the pH reaches ~7.2 as it will fall rapidly thereafter and if you overshoot you must start again.
- Add DTT or 2-mercaptoethanol immediately prior to using:ie. to 1ml SDS SB add 50Ál 1M DTT (from frozen stock) or 40Ál 2-ME solution.
  1. Loading sample buffer with bromophenol blue
- 20 ml 1 x SDS sample buffer
- 2 ml bromophenol blue (1mg/ml) 0.1% in DDW. Aliquot 1ml and store 4░C.
  1. 10x SDS running buffer pH ~8.3 (2 Lt total)
Trizma base   60.6 g
Glycine   288.0 g
SDS   20 g
DDW to   2 Lt
- NOTE: Do not pH
  1. Lower gel buffer (LGB) pH 8.8 (500 ml total)
Trizma base 91 g
SDS 2 g
DDW 450 ml
Adjust pH using concentrated HCl
Add DDW to give 500 ml total volume
  1. Upper gel buffer (stacking gel) pH 6.8 (250 ml total)
Trizma base 15 g
SDS 1 g
DDW 200 ml
Adjust pH using concentrated HCl
Add DDW to give 250 ml total volume
  1. Coomassie blue (2 L)
2 g Coomassie brilliant blue powder (BioRad)
1 L methanol
200 ml acetic acid
800 ml DDW
Stir for a minimum 2 hrs
Filter through Whatman filter discs
  1. Novex Pre-Stained Markers
Use these standards. They should be heated to 100 degrees for 5 min. prior to dividing into 20Ál aliquots for storage at 4 degrees. Load 7Ál/well for a 15 well comb, and 10Ál/well for a 10 well comb. (on Bio-Rad mini gel)Reagents for Protein Mini-Gels1 mini gel
% 7.0 10 12 15 20
LGB 1.34 ml 1.34 ml 1.34 ml 1.34 ml 1.34 ml
DDW 2.5 ml 2.0 ml 1.65 ml 1.0 ml 0.35 ml
30% 1.16 ml 1.66 ml 2.0 ml 2.5 ml 3.35 ml
APS 25% 10 Ál 10 Ál 10 Ál 10 Ál 10 Ál
TEMED 10 Ál 10 Ál 10 Ál 10 Ál 10 Ál
2 mini gels
% 7.0 10 12 15 20
LGB 2.67 ml 2.67ml 2.67 ml 2.67 ml 2.67 ml
DDW 5.00 ml 4.00 ml 3.33 ml 2.0 ml 0.7 ml
30% 2.33 ml 3.33 ml 4.0 ml 5.0 ml 6.7 ml
APS 25% 20 Ál 20 Ál 20 Ál 20 Ál 20 Ál
TEMED 20 Ál 20 Ál 20 Ál 20 Ál 20 Ál
3 mini gels
% 7.0 10 12 15 20
LGB 4.0 ml 4.0 ml 4.0 ml 4.0 ml 4.0 ml
DDW 7.5 ml 6.0 ml 5.0 ml 3.0 ml 1.0 ml
30% 3.5 ml 5.0 ml 6.0 ml 8.0 ml 10.0 ml
APS 25% 30 Ál 30 Ál 30 Ál 30 Ál 30 Ál
TEMED 30 Ál 30 Ál 30 Ál 30 Ál 30 Ál
4 mini gels
% 7.0 10 12 15 20
LGB 5.4 ml 5.4 ml 5.4 ml 5.4 ml 5.4 ml
DDW 10.00 ml 8.00 ml 6.66 ml 4.0 ml 1.34 ml
30% 4.66 ml 6.66 ml 8.0 ml 10.0 ml 13.7 ml
APS 25% 40 Ál 40 Ál 40 Ál 40 Ál 40 Ál
TEMED 40 Ál 40 Ál 40 Ál 40 Ál 40 Ál
Stacking gels
  1 gel 2 gels 3 gels 4 gels
UGB 0.61 ml 1.25 ml 1.86 ml 2.5 ml
DDW 1.5 ml 3.0 ml 4.5 ml 6.0 ml
30% 0.4 ml 0.8 ml 1.2 ml 1.6.0 ml
APS 25% 5 Ál 10 Ál 15 Ál 20 Ál
TEMED 5 Ál 10 Ál 15 Ál 20 Ál
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998