Source: Contributed by Sharad Purohit, Paller's Lab
Date Added: Tue May 14 2002
Date Modified: Thu Apr 29 2004
Sodium acetate Buffer (200mM, pH 5.5): Prepare a 200 mM solution of sodium acetate by disolving 12.0 gam of sodium acetate trihydrate in 440 ml of dw. Prepare a 200mM solution of acetic acid by dissolving 690ul of glacial acetic acid to 60 ml Dw. Mix the two solution and check pH
PBS pH 7.2: dissolve 575 mg disodium phosphate , 100 mg of sodium dihydrogen phosphate and 800 mg sodium chloride in 500 ml Dw.
Sodium acetate buffer/5mM EDTA: dissolve 1.14 g of EDTA (Tetra sodium salt ) in 100 ml of 200 mM sodium acetate buffer and dilute to 200 ml with Dw
10mM sodium meta periodate solution in 100mM sodium acetate/5mM EDTA
Biotin Hydrazide: Prepare a stock solution of 1mg/ml in DMF. Working solution is 100ug/ml in 100mM Sodium acetate buffer containing 5 mM EDTA pH5.5. Prepare fresh every time
Blocking Solution:; 5% gelatin in TBS-T.
Color development reagents
Wash the blot extensively with PBS to remove traces of tris (as tris interferes with biotinylation reaction).
Immerse the blot in 10ml of 10mM sodium periodate solution and incubate in dark for 25 minutes.
Wash membrane 3 times with PBS for 10 minutes each was at room temperature with gentle agitation.
Prepare the working biotinylation solution just before use and incubate the membrane in thei solution for 90 minutes at room temperature with gentle agitation
Wash the membrane 5 times with TBS-T (TBS-T will no longer interefere with the process from this stage onwards) for 20 minutes with gentle agitation
Block the membrane in blocking solution for 1 hour at 37 deg C.
Wash membrane with TBS-T and add the secondary antibody conjugated with Alkaline Phosphatase or HRP for 1 hour.
Wash the membrane with TBS-T.
Develop blot with the devlopment reagent of the choice till you get a desired contrast.
Tris, 2-mercaptoethanol, ammonium ions, DTT, monosaccharides, sodium borohydride transtion metals and urea are the interfereing compunds. Hence avoid their use before the biotinylation reaction.