Laboratory of P.J. Hansen,Dept. of Animal Sciences, University of Florida http://www.animal.ufl.edu/hansen/protocols/blotting.prt.htm
REAGENTS
ECL Western blotting kit (Amersham Life Science; cat# RPN2108): contains second antibodies for both mouse and rabbit, substrate and milk blocker (the milk blocker is not normally used when using ruminant samples).
Hybond ECL nitrocellulose membrane (Amersham Life Science; cat # RPN2020D)
Kodak X-OMAT (XAR-5, 18X24 cm; cat # 8532665)
10X TBS
12.11 g Tris-base (100 mM)
87.66 g NaCl (1500 mM)
1 liter dd H2O
Adjust pH= 7.6
Washing buffer (TBS-T): 100 ml 10X TBS + 900 ml dd H2O + 1 ml Tween-20.
Blocking buffer: TBS-T + 1.5% gelatin
Incubation buffer I (for first antibody): TBS + 1.5% gelatin
Incubation buffer II (for second antibody) = blocking buffer (i.e., TBS-T + 1.5% gelatin)
PROCEDURES
1- Immediately after removal from the blotting apparatus, place membranes into blocking buffer for 2 hours. A small plastic gel box is a suitable container. This and all other incubation steps are performed at room temperature and in the rocker platform.
2- Wash membrane in washing buffer: rinse 2 times very briefly, incubate for 15 minutes, then repeat 2 x at 5 minutes each. Use a lot of buffer.
3- Transfer the membrane to a lid of 96-well microtiter plate or similar low volume container. Incubate with first antibody using recommenced dilution in TBS + 1.5% gelatin during 2 hours. Approximately 10 ml of diluted antibody showed to be sufficient.
4- Transfer the membrane into gel box and repeat step # 2.
5- Transfer the membrane back to the small container and incubate with anti-mouse or anti-rabbit IgG horseadish peroxidase diluted 1:8000 in TBS-T + 1.5% gelatin for 1 hour.
6- Repeat step # 4 (wash)
7- Prepare ECL solution for detection: mix equal volume of ECL reagent 1 and 2, (1:1) with final volume regarding of 0.125 ml/cm2 (for a mini gel- 4 ml of each reagent).
8- Remove excess buffer from the membrane by draining the membrane over a piece of folded kimwipe paper and briefly touching the edge of the membrane to the paper.
9- Add ECL solution and incubate for 1 minute.
10- Repeat step # 8 ( drain excess of substrate)
11- Place membrane down on seran wrap, remove bubbles and wrap membrane completely.
Avoid excess amounts of seran wrap.
12- Tape membrane to the inside film cassette.
13- In the dark, add 1 sheet of x-ray film to the cassette. Expose membrane to film. It will probably be necessary to do several different exposures to find out the best exposure.
14- Develop the film.
