SDS-PAGE

General: Separation of protein by size, under reducing or nonreducing conditions. The percentage of acrylamide used depends on the size of protein.

Solutions:

30% Acrylamide for SDS gels (store at 40C) 500mL Buffer
- 29.2% Acrylamide -146g
- 0.85Bisacrylamide -4g
- solvent is ddH2O
Note-Filter through Whatman #1 paper

Upper gel buffer, pH 6.8 (store at 40C) 500mL
- 0.5 M Tris base -30.28g
- 0.4% SDS -2 g

Note-pH before adding SDS
- bring to Ph 6.8 with conc. HCl(about 8mL) The pH gradient is very steep near pH 7(don't back titrate)

Lower gel buffer, pH 8.8 (store at 4°C) 500 mL
-1.5 M Tris-base -90.82 g
-0.4 % SDS -2 g

Note-pH before adding SDS
bring to pH 8.8 with conc. HCl(about 8mL) (don't back titrate)

SDS sample buffer pH 6.8 (store at 40C) 100mL 1x 4x
-62.5 mM Tris base -6.25mL 1M 12.5 2M
-10%(v/v) glycerol -10mL 40mL
-5%(v/v)b-mercaptoethanol -5mL 20mL
-2.3%SDS -2.3g 9.2g
-10% v/v Bromphenol Blue -10mL 0.01g
Note-pH before adding SDS

Running buffer(1x) (do not pH, store at RT) 1L 10x
-25mM Tris Base -30.28g
-192mM glycine -144.13g
-o.1%SDS -10g

Bromphenol Blue (store at RT) 50mL
-0.1%bromphenol blue -0.05g
-10mM Tris pH 6.8 -0.5mL 1M stock

Coomassie Blue (conc.) 500mL
-0.1%Sigma brilliant blue R -0.5g
-20% Methanol -100mL
-7% Acetic Acid -35mL

Destain (store at RT) 4L
-20% Methanol -800mL
-7.5% acetic acid -300mL
-H2O -2.9l

Running Gel Solution 10% 7.5% 12.5% 15%
-ddH2O -6.7mL -8.0mL -5.3mL -4mL
-lower gel buffer -4mL -same -same -same
-30% Acrylamide -5.3mL -4mL -6.7mL -8mL
then add
-10% NH4 persulfate -25mL -same -same -same
-TEMED -12.5mL -same -same -same
Note: To make other % gels only change the amount of acrylamide and ddH2O. For X% gels:
-vol.(mLs) of 30% Acrylamide=X(desired gel %)x 16mL/.3
-vol.(mLs) of ddH2O=10mLs-vol.of 30%Acrylamide
-For Minigel make 8mLs total with the same proportions

Stacking Gel Solution (enough for 2 gels)
-2.5mL upper gel buffer
-5.9mL ddH2O
-1.6mL 30% Acrylamide
then add
-30mL 10% Amonium persulfate
-10mL TEMED
Note: The stacking gel does not change with changes in the percent of the running gel.