Laemmli Gels

Laemmli: Nature 227, 689 (1970)

 

  1. Assemble the gel mould, mix the solutions below in order and pour gel immediately after adding APS.

 

Running Gel

 

18%

15%

12.5%

11%

10%

8%

7.5%

6%

A

18.0

15.0

12.5

11.0

10.0

8.0

7.5

6.0

B

7.5

7.5

7.5

7.5

7.5

7.5

7.5

7.5

H2O

4.3

7.4

9.9

11.3

12.4

14.4

14.8

16.4

Temed

10ul

10ul

10ul

10ul

10ul

10ul

10ul

10ul

APS

200ul

200ul

200ul

200ul

200ul

200ul

200ul

200ul

Make 10% APS fresh each day this is the initiator and should be added immediately before pouring - old APS may appear to work but the quality of the crosslinking will be compromised. Temed speeds up the rate of but cannot start the polymerization. The rate of polymerization is also affected by temperature.

2) Overlay gel with water saturated isobutanol. Polymerize for ~ 1hr. Gels can then be used immediately or stored for 2-3 days covered in saran wrap.

Optional: prerun this gel at 40mA for 1hr this sharpens the bands but compresses them too such that for example a 7.5% gel will run like a 10% gel.

Stacking Gel

Do not pour until 1hr before you are ready to run the gel or you will get pH diffusion between the gels resulting in fuzzy bands. Mix solutions pour on top of stack gel and insert the comb. Once the gel is set remove the comb and immediately rinse out the pockets to get rid of dribbles of acrylamide that set and collapse into the pocket. Assemble gel apparatus, add running buffer and load samples. Insert into chamber- make certain that the lower electrode is covered by buffer. Boil samples to fully solubilize the proteins for 5 mins then spin to remove insoluble debris for 5 mins.

 

3%

3.6%

3.8%

H2O

6.5

6.3

6.2

A

1.0

1.2

1.3

C

2.5

2.5

2.5

Temed

10

10

10

APS

100

100

100

Run gels for 120mA/hrs i.e. 3hrs at 40 mA or O/N at 10-12 mA

The best gels are achieved when the sample volumes and protein and salt content are approximately equal. Smaller sample vols are better. If using large vols make the stack gel bigger.

Laemmli Gels

Buffers: Filter buffer A through 3MM Whatmans and store 4C; Store B and C and RB at RT; store sample buffer in small aliquots frozen and be careful that the DTT is fresh.

Buffer A Acrylamide: Bis (30:0.8)

300 g Acrylamide

8 g Bis

1L H2O

Buffer B 1.5 M Tris / 0.4%SDS

90.85 g Tris pH 8.8

2 g SDS

500 ml H2O

Buffer C Tris 0.5M / 0.4% SDS

6.06 g Tris pH 6.8

0.4 g SDS

100 ml H2O

10X Running Buffer (1 X = 25mM Tris; 190mM glycine 0.2% SDS)

28 g Tris

20 g SDS (or 100 mls of 20% soln) - could use only 10g/L

143 g Glycine

1L H2O

Sample Buffer (2X)

100 mM Tris pH 6.8

4% SDS

200 mM DTT

20 % glycerol

0.1% Bromophenol Blue - could use 0.01%

Other recipes use: 10mM phosphate pH 7.5 (2mls of 100mM NaH2PO4 + 8mls of 100mM Na2HPO4.2H2O)-considered to be better for membrane proteins; 5% SDS; 10% Mercaptoethanol

Stain (20X) add 25ml/500ml of destain or fix (Old recipe uses 1g/100mls and 12/500)

5g Coomassie Brilliant Blue R

90 ml methanol

10 ml acetic acid

Destain

45% methanol

10% acetic acid

Fix / 1L - do not use with gels that will be fluorographed but gives a fast stain after only 30mins.

12g TCA

200 ml methanol

70 ml acetic acid