1) 1.5 M TrisHCl pH8.3 + 0.4% SDS (adjust pH before you add the SDS).
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
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.
Number of minigels 5 8 5 8 5
Stock solution 1 (1.5M)
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.
Number of minigels 2 5 8
Stock solution 3 (0.5M)
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
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”.