Hi

1. Why cant you try in PVDF membrane and use the recommended blocking solution.

2. Try to reduce the antibody concentration

3. Reduce the developing solution exposure

What is your washing procedure? How much time your blocking your blot?

Block in 5% BSA in TBST for 1 H, then give two mild washes with in a 5 min.

Follow the probing, then while washing step give a 3 washes (Each wash 7 min) for primary AB as well as Secondary AB.

Make sure your detection reagent is not expiry. Try using fresh (new) detection reagent. Mix detection reagent A and B only before use (western blotting detection reagent kit). Exposing detection reagent to light causes solution to degrade over a time.

Sarwareddy Kartik Kumar I block for 1 hr. I do not wash after doing the blocking, just proceed to incubate with the primary antibody overnight. I do 3 5 min washes of TBST 1x, then secondary for 1 hr, then 3 5 min washes of TBST again.

Dear Raquel, appearance of ghost or hollow bands are in general due to increased concentration and an increased incubation time of enzyme-labelled antibodies (like 2ndry antibodies), which uses up almost the entire substrate far before being detected on gel doc systems.

Further, if you are using nitrocellulose membrane so be careful not to apply any excess transfer time, as loss of proteins is very common in such membranes towards the adjacent filter papers or into the transfer buffer solution. So it is better to switch to PVDF membranes though they have higher backgrounds than the NC membranes. NC membranes are only good for smaller proteins.

Improper washing of membranes, drying of membranes, precipitation of proteins are some of the factors contributing to an increased background. Never load your gel with warm samples like immediately after the heat denaturation. Cool down your samples on ice first then load the wells.

If you use too much exposure to ECL then you can wash the membrane after this exposure, pour again fersh ECL, rinse off excess solution from membrane and perform reimaging. It does work often.

Sincerely....

Here a link to our comprehensive western blotting guide https://www.phosphosolutions.com/protocols-western-blot/