PAGE-SDS Laemmli Protocol

Stock Solutions

1) 1.5 M TrisHCl pH8.3 + 0.4% SDS (adjust pH before you add the SDS). Keep RT.
2) 30% Acrylamide 0.8% Methylene bis Acrylamide. Keep 4C.
3) 0.5 M TrisHCl pH6.8+ 0.4% SDS (adjust pH before you add the SDS). Keep RT.
4) 10% Ammonium Persulfate (APS). Keep 4C max. 1 month.

Running Buffer x5
Trizma         0.125M          15.1gr
Glycine        0.96M            72.0gr
SDS             0.5%                5.0gr
H2O up to                           1 liter
 Keep at RT

Dissolving Buffer x5
 Glycerol                       5ml
 SDS                              1gr
Beta Mercaptoethanol  2.56ml
Stock solut. 3 (0.5M)   2.13ml
Bromo Phenol Blue      traces
Keep in aliquots of 1ml at –20C

An alternative formulation (4xSB with DTT) can be found here.

Separating Gel

% Acrylamide                            10%        10%        12%        12%       15%
Number of minigels                      5             8             5             8             5

Stock solution 1 (1.5M)            7.0ml       10.5ml     7.0ml     10.5ml     7.0ml
Stock solution 2 (Acryl)           9.3ml        13.9ml   11.3ml     16.9ml   13.9ml
H2O                                         12.3ml       18.4ml    9.3ml      13.9ml     6.3ml
10% APS (stock sol.4)             100ul         150ul     100ul       150ul     100ul
TEMED                                     23ul           35ul       23ul         35ul       23ul

Add TEMED and APS at the end. Gently swirl the flask to mix, being careful not to generate bubbles. Pipette the solution to a level of 4cm from the top. Add 0.3ml of n-buthanol. A very sharp liquid interface will be visible within 10-20min. Let  the gel polymerize for another hour at least. Rinse the surface of the gel with H2O before pouring the stacking gel.

Stacking Gel

Number of minigels                        2            5           8

Stock solution 3 (0.5M)            2.5ml      4.0ml      5.2ml
Stock solution 2 (Acryl)            1.0ml      1.5ml      2.0ml
H2O                                           6.4ml      9.6ml     12.8ml
10% APS                                   100ul      150ul      200ul
TEMED                                      10ul         15ul       20ul

Fill each sandwich with stacking gel solution and insert a comb into each place taking care not to trap any bubbles bellow the teeth. The gel should fully polymerized after 1-2 hours. Keep gels no more than 2 weeks cover with a wrap nylon.

Staining Solution                                            Destaining Solution

Methanol CP                       50%                 Methanol CP                 15%
Acetic Acid CP                   10%                 Acetic Acid CP             10%
Coomasie Brilliant Blue R  0.25%

Keep flasks in dark at RT

Stain ON at RT or put gel with staining solution 8 sec in microwave at position 10, and then shake for another 15min at RT. Wash with water 2-3 times and destain in the presence of a “sfog”.