SDS-PAGE Gels old (long) protocol

SDS Gel electrophoresis

gels with SDS (makes 2 baby gels; 24 wells total))

Separating Gel (goes on the Bottom of the gel):
40% (19:1) Acrylamide:Bis Acrylamide solution 3ml
1M Tris Buffer pH 8.9 4.68ml
ddH20 4.2ml
10% SDS solution 125ul
total volume 12ml
pour 4ml per gel (up to bottom of green bar)

1. Mix and add 50ul of fresh ammonium persulfate (0.05g/500ul of water) and 8ul of Temed. (The more Temed you add, the faster the polymerization)
2. Pour quickly using small beaker
3. Layer 2-methyl-1-propanol (iso-butanol) on top or plain ethanol or just water (all work well for us)
4. Leave at room temperature for >30min to polymerize
5. Wash out isobutanol with distilled water

Stacking Gel (goes on the top of the gel where the combs are):
40% (19:1) Acrylamide:Bis Acrylamide solution 0.735ml
1M Tris Buffer pH 6.8 0.875ml
ddH20 5.24ml
10% SDS solution 75ul
total volume 6.93 ml
pour 2ml per gel (leave 3mm gap to top of small plate)

1. Mix and then add 50ul of fresh ammonium persulfate (0.05g/500ul of water) and 5ul of Temed.
2. Pour quickly using small beaker
3. Place combs in, clean up any spillage
3. Leave at room temperature for >20min to polymerize
4. If not using immediately, pull combs out and wash wells with water, remove plates from rack, wrap in saran wrap, label with name and date
5. If using immediately, put into gel box so that small plate faces the core of the box; or 1 gel facing in and the buffer dam on the other side)
6. Lock the plates in, pour buffer into the core of the box so that it goes over the combs and into the bas of the box
7. Remove combs very carefully pulling straight up, the wells break easily and can "fall" into each other
8. Use a pipette tip to straight any out of line wells


2x Sample buffer choice #1
Tris Buffer pH 6.7 2.5ml
Glycerol 2ml
water 3.3ml
Filter through a 0.45uM filter and then add
10%SDS 2ml
2.5% Bromophenol blue 2.5mg
total volume 10ml

2x Sample buffer choice #2
Tris Buffer pH 6.7 10ml
10% SDS 15ml
80% Glycerol 6.25ml
2.5% Bromophenol blue 0.5ml
water 2ml
total volume 40ml

1. Store sample buffer at RT; (before use add 1 part 1M DTT to 4 parts sample buffer #2 (not necessary for #1))
2. Dilute samples with sample buffer 1:1, boil at 100degrees for 2 min.
3. Run baby gel at 8milliamps until dye has reached the separating gel then at 25milliamps until dye has reached the bottom of the gel


10X SDS Running buffer
Glycine 1134g
Tris base 240g
Add to 6 Liters of ddH20 while stirring
After it is dissolved bring to final volume of 8L
check ph, bring to 8.8, store at RT

1XSDS Running Buffer
Make fresh from 10X (1L of 10x into 9L of water)
Add 20% SDS to give final concentration of 0.1% (50ml into 10L)