SDS-Polyacrylamide Gel Electrophoresis
Detection of biotinylated 2'Ab with Vectastain ABC-AP.
									
SDS-Polyacrylamide Gel Electrophoresis (PAGE)
Denatured with SDS.

1.  Separating gel: 15ml
	15% PA gel is as follows:
	7.5ml of 30% acrylamide/0.8% bisacrylamide
	3.75ml of 4x Tris-Cl/SDS, pH 8.8
	3.75ml ddH2O
	0.05ml 10% ammonium persulfate (APS)  Just prior to use.
	0.01ml TEMED Just prior to use.

2. Stacking gel: 5ml
	0.65ml of 30%acrylamide/0.8% bisacrylamide
	1.25ml of 4x Tris-Cl/SDS, pH 6.8
	3.05ml of ddH2O
	0.025ml of 10% APS
	0.005ml of TEMED

3. Clean two glass plates (14cm x 14cm). place spacers (0.75cm) so just stick out, clamp, place on parafilm into gel apparatus.
4. Add separating gel to space at one side and move across space to get everywhere. 
NO bubbles. Use a 10ml pipet.
5. Top with N-butanol saturated with H2O. Let harden for 1 hour.
6. Rinse off N-butanol 3-4x with ddH2O. 
7. Add stacking gel all the way up to rid any bubbles.
8. Add comb then polymerize for 1 hour. Remove comb and rines with running buffer.
9. Set up apparatus for 2 gels to balance. Use appropriate clamps.
10. Place into apparatus. Cover with running buffer (1X SDS). Install cover.
11. Boil samples and molecular weight marker for 5 minutes then spin down.
12. With a glass syringe, add 30-40 ul of Lammli buffer and 2 ul bromophenol blue to first sample, mix, then add to gel. Repeat with other samples. 
Add buffer to all lanes so gel does not bend.
	Note: add 5ul of marker to each end of the gel.
	Note: proteins move from cathode to anode (- to +).

13. Wash syringe 3-4x between each use.
14. Run gel at 10mA with running water cooler hooked  up. Increase to 15mA when dye reaches separating gel. 
15. When dye reaches bottom turn off power, remove apparatus, take off top glass, cut off bottom left corner and stain with Silver, Coomassie blue according to instructions or proceed to Western blot detection of proteins.

go to running a Western blot