Sean Lauber:SDS PAGE Protein Electrophoresis

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1) Prepare the necessary components for the electrophoresis apparatus and wash the glass plates with 70% EtOH

2) Prepare the resolving gel (add APS and TEMED last):

 Prepare the gel mix as shown on the recipe sheet (7.5 ml/gel), ADD THE TEMED LAST…often 8% acrylamide is enough, but for smaller proteins you may want a higher %
 Pour the resolving gel between the glass plates 
 Add a bit of 95% EtOH to the top of the gel to eliminate bubbles and flatten the gel solution
 Once the gel has polymerized (about 20 min), decant the EtOH and wash 3 times with water (use a squirt bottle and ensure all bubbles are gone)

3) Prepare the stacking gel (add APS and TEMED last):

 Prepare the stacking gel as shown on the recipe sheet (about 3 ml/gel)
 Pour the stacking gel on top of the resolving gel 
 Insert a clean comb (10 or 15 well) into the stacking gel, being sure no bubbles form below the wells
 Once the gel has polymerized (about 20 min), carefully remove the comb

4) Prepare the samples:

 If you're using a 10 well comb, each well can hold 30 ml, for 15 well, 20 ml
 You want to load about 8-15 ug protein (from Bradford) per well
     To prepare the 5X sample buffer…
       SDS	                1.5 g
       1 M Tris pH 6.8	        3.75 ml
       Bromophenol blue	0.015g
       B-mercaptoethanol  	0.75 ml
       Glycerol	        7.5 ml
       ddH2O	                3.0 ml
     Add water until 15 ml in volume and mix well, store in 1 ml aliquots at -20*C	
 Boil samples for 5 min

5) Pour the running buffer into the apparatus, aiming on top of the gel to help dislodge any tiny bits of gel debris

 For 1 L running buffer (enough for one running chamber):
   10X Tris	100 ml
   10X Glycine	100 ml
   10% SDS	10 ml
   ddH2O	790 ml

6) Prepare and load the molecular weight ladder (10-170 kDa range) - usually use 3.5 ul (FroggaBio), but may use more especially if you're going to strip the blot later (use 5 in this case)

7) Load your samples

8) Run the gel so the samples start stacking at 90V (about 15 min), then once the band has entered the resolving gel (the band will now be fully stacked) increase the voltage to 120 V and run for 1 hour


Image:SDS_PAGE_gel_formulations.png

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