High background

Blocking of non-specific binding might be absent or insufficient

Increase the blocking incubation period and consider changing the blocking agent, we recommend 3–5% non-fat dry milk, BSA, or normal serum for 1 h at room temperature. These can be included in the antibody buffers as well.

The primary antibody concentration may be too high

Titrate the antibody to the optimal concentration. Incubate for longer but in more dilute antibody (a slow but targeted binding is best).

The incubation temperature may be too high

Incubate membrane at 4°C.

The secondary antibody may be binding non-specifically or reacting with the blocking reagent

Run a secondary control without the primary antibody.

Cross-reactivity between the blocking agent and primary or secondary antibody

Add a mild detergent such as Tween 20 to the incubation and washing buffer.

For phospho-specific antibodies: milk contains casein which is a phosphor-protein; the phospho-specific antibody will detect casein present in the milk causing high background. Use BSA as a blocking reagent instead of milk

Your choice of membrane may give high background

Nitrocellulose membranes are considered to give less background than PVDF.

The membrane has dried out.

Care should be taken to prevent the membrane from drying out during incubation

Hi Robert, let me get this straight.

1) you have made affinity purified antibodies against a protein from a parasitic nematode.

2) you did westerns using worm extracts and probing using the Ab previously mentioned.

3) Pre-immune serum is reacting to the protein and immune serum is not.

I am really confused. If you have an immuno-purified antibody already in hand, why are you using pre-immune and immune serum to probe the blot instead of normal blockers (BSA or milk)?

You said that you had an affinity purified antibody, so why on earth are you not using it instead of the pre and post immune serum.? There might be factors in the serum blocking the epitopes

Do you really have a purified antibody or just a serum sample that shows some positivity against the antigen? 

Please explain.  

@Steingrimur Stefansson

Here are answers to your questions

1. I have affinity purified antibodies against the antigen

2. I used the affinity purified antibodies against worm extracts on WBs, it doesn't recognise a specific band and the pre-immune reacts too.

Actually we want to do immunolocalisation that why we are probing with both the purified antibodies and preimmune serum.

Is that possible that the host animal which you used for immunization might already have this parasitic nematode or similar parasite in their gastro-intestinal tract ? Sometimes, parasites might induce some antibodies in the "healthy" host animals. That would explain your observation with pre-immune serum. 

Hi Robert, so you have an affinity purified antibody against an antigen, but it doesn't recognize the antigen on WB.

I assume you made an antibody against a purified antigen. Does the Ab recognize the antigen when the purified antigen is run alone on WB? 

If the purified Ab does not recognize the purified antigen it was made against on WB, then there is something seriously wrong.

If you are working with monoclonals, I can understand that epitopes can be hidden during blotting, but at least you can do is IP.

If you have an affinity purified polyclonal, there is no reason why it would not work on WB. Unless there is something seriously wrong. 

@Areeful,

Here are the answers to yours questions:

1. I am blocking with with BSA for 2hours at room temperature

2. I used nitrocellulose

3. I used primary antibody at 1:1000 and secondary antibody at 1:5000

Western blot is a highly underestimated technology that goes wrong most of the time. Only well trained operators know how to run western blots without problems. Before we jump to conclusions, you first have to assess whether the antibody is wrong, or the western blot procedure went wrong.

Check the antibody: Dot purified protein at different amounts on a NC membrane under native conditions. Do the same after the protein has been reduced and denatured. Now probe this dot blot with your purified antibody at your optimal dilutions (start at 1ug IgG/ml and try 0.3 and 0.1ug/ml). Do you get signals consistent in strength with the amount of protein in the dots and with the dilutions of the antibody? If not, stop using this antibody and address your immunization protocol.

Check the Western blot procedure: Run two identical western blots with purified protein and with worm extracts. One blot is exposed to the purified antibody and the other is not. Both are labelled with the secondary and being stained side-by-side. If the membrane that has not been exposed to the primary shows bands, you have your answer: Your secondary antibody is rubbish.