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1) Dot primary antibody positive controls on edge of filter. Let this edge thoroughly dry (keep remainder of filter wet between Whatman 3MM paper).
2) PBS wash (100 ml each) 5 minutes X 2.
3) Block 60 minutes at 370C in shaking water bath with PBS/5%BSA/1-t GOAT SERUM* ( * Note: use normal serum from same species in which the secondary antibody was made)
PBS 99 ml
BSA (5%) 5 grams
Goat Serum (1%) 1 ml
100 ml
4) Incubate filter with 10 ml of primary for 2 hours at room temperature (this may filter antibody in a sealed bag, depend on antibody) on a rocker. The concentration of antibody will need to be determined empirically, although as guidelines I would use approx. 5ug/ml of a monoclonal and a 1:300 dilution of a polyclonal antibody.
5) PBS wash (100 ml each) 5 minutes X 3.
6) Incubate filter with secondary antibody (Biorad, dilute 1:3000) for 60 minutes at RT on shaker.
PBS 99 ml
Goat Serum (1%) 1ml
Secondary antibody 33 microliters
100 ml
7) PBS wash (100 ml each) 5 minutes X 3.
8) Prepare BCIP/NBT and use carbonate buf f er as per standard Biorad Kit instructions (prepare just prior to use):
Carbonate Buffer 0.1 M NaHCO3, 1-0 mM MgCl2, pH 9.8)
To make 1L: add 8.40 grams NAHCOI, 0.203 grams MgCl2 6H2?
to 900 ml distilled H20. Adjust to pH 9 . 8 with NAOH, and bring volume up to 1L.
i) NBT Dissolve 30 mg of BCIP in 1 ml of 70% DMF (N,Ndimethylformamide) (0.7 ml DMF and 0.3 ml water)
ii) BCIP Dissolve 15 mg of BCIP in 1 ml of DMF.
Just prior to use, add NBT and BCIP stock solutions to 100 ml of room temperature carbonate buffer. Mix. Immediately add to blot. Put on shaker and observe band development.
9) When development is adequate, quench reaction by removing development solution and wash blot several times with water. Wrap blot in saran wrap and take photograph.
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