Laemmli Gels
Laemmli: Nature 227, 689 (1970)
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