ECL reagent: homegrown version

Advantages: almost no cost; stable/reproducible, stored in frozen aliquots

To 10 mls of 100 mM Tris pH 8.5 (RT), add

  1. 50 mls of luminol (warm to redissolve)
  2. 22 mls of coumaric acid (warm to redissolve)
  3. 3 mls of H2O2 (fresh < 6 months)

Pour onto blot for 1 minute and process as normal (10 mls enough for 100 cm2)

Stock luminol: 250 mM 3-aminopthalhydrazide (Fluka #09253); 266 mgs in 6 mls DMSO; store frozen in 60 mls aliquots.

Stock coumaric acid: 90 mM coumaric acid (Sigma C9008): 38 mgs in 2.5 mls DMSO; store frozen in 25 ml aliquots.

 

 

Stripping blots:

Most antibodies should be removed by heat and SDS:

100 mls

b-ME 100 mM 0.71 mls 14.1 M stock from bottle

SDS 2% 20 mls 10% stock

62.5 mM Tris, pH 6.8 12.5 mls SDS-PAGE upper gel buffer

Wash blot well with aq, including warm water, add stripping solution warmed to 50oC in closed container, incubate 30 mins, decant with large volume of running water, wash again with aq, and put into Blotto to reblot. Would recommend putting back into ECL to check efficiency of strip, and repeating the secondary control to check for background. Note foreground goes down with every strip, and background comes up.