Biotechnology and Bioinformatics (1+1)

Immunological detection of protein

1. After transfer, the membrane is washed in blocking solution ( 5 % milk powder in 1 X PBS) for 20 min . The solution is discarded and this washing is repeated for 2 -4 times .

( Note: Blocking the membrane prevents non-specific background binding of the primary and / or secondary antibodies to the membrane (which has a high capacity at binding proteins and therefore antibodies).

2. Then primary antibody [Anti hemolysin rabbit serum] (1 : 500) diluted in 30 ml of blockig solution is added . The membrane and antibody mixture are rocked gently for 45 min- Ihr.

3. After incubation the solution is discarded and the membrane is washed with IXPBS for three times.

4. Then secondary antibody (Alkaline phosphatase conjugated goat anti-rabbit antibodies) diluted (l :1000) in 30 ml of the blocking solution is added . The membrane and antibody mixture are rocked gently for Ihr .

5. After incubation the solution is discarded and the membrane is washed with IX PBS for three times.

6.The membrane is developed in dark by adding about 20 ml of developing solution (BCIPINBT solution- Generally, BCIPINBT Phosphatase Substrate deposits a permanent, dark purple stain on membrane sites bearing phosphatase)