it seems that there is specificity problem with your antibody/serum. Do a western bolt to check if that is the case, In your western blotting, your antibody/serum should only recognize your target protein but not any other cellular proteins. If it recognizes both, you need to do an immunoprecipitation of your antibody with cellular extracts of your untransfected cells to clean the antibody/serum, and then use it for your dot blotting.

You should first test the anti-serum with a positive control and a negative control, before you test your transfected and untransfected cells. No antiserum is 100% specific, so there will always be background. You need to know how much of your viral protein is needed to give a signal above background. Is it 1 nanogram, 10 nanogram, or 100 nanogram? If your serum detects 1 nanogram, that's good and you should be able to see it in your transfected cells if the plasmid is correct and there is no mistake in your expression construct. If you need 10 nanograms, then the antiserum is poor. If you need 100 nanograms, then the antiserum is very very poor because you can see 100 nanograms of protein on coomassie stained gels. If you don't have positive and negative controls, then make them first, otherwise you cannot distinguish between transfection problems or dot-blot problems. I agree with Huanting that a western blot from a proper SDS-PAGE of cell lysates (trasfected and untransfected next to each other) is the best way to check because you will be able to verify the viral protein size by molecular weight and you will see other sized bands that could give background signals. If you have lots of background bands, but you see a specific band appear in the transfected sample, then you can improve your antiserum by immuno-absorbtion (using a solid state library of proteins from untransfected cells, i.e. cross-linked to Sepharose beads) to subtract unwanted antibodies in your serum. Before you do any of that, you must be able to detect a positive control.

Hi Asia, are you using an HRP labeled secondary for ECL detection? Do you have to expose the film for > 10 sec for a signal?

If so, then the quality of the HRP secondary is not good and it will influence the background on whatever immunoassay you do, whether it be dot blot or western.

Thank you all for your suggestions......

I agree with both Liu and Denecke. I have some suggestions.

How is your antisera produced?

if you have used cell culture produced virus  for immunization of animals to produce sera, expect antibodies against cellular proteins. That is not the end of the story- expect antibodies against serum proteins. in fact expect more antibodies against serum proteins than the cellular proteins.  

Now you have more antibodies against cellular proteins and serum proteins than your virus proteins.

Best way is to purify the virus by density gradient centrifugation (ultracentrifugation) for any immunization protocols to produce antisera for western/ELISA.

you test your antisera with positive and negative controls following above suggestions.

To get rid of non-specific antibodies we follow a simple procedure instead of using  immunoabsorption or immunoprecipitation.

Resuspend cells  along with media freeze thaw three times and centrifuge at 10000g to remove cell debris. Now, your lysate consist both cellular and serum proteins. Add this lysate to your antisera at 1:1 ratio and teat for non specific antibodies by western or ELISA. if needed you can adjust the ratio. in our hands 1:1 works for most of the times. 

Here we are not depleting the antibodies in the serum. We are creating competition between non-specific protein in the blot (fixed) and in the serum (in liquid). If excess nonspecific protein is available  in the liquid phase most of the nonspecific antibodies will stay in liquid phase and washed away during washing. increased amount of nonspecific protein in the liquid phase will reduce the background.

Best

Thank you Panduranga.

Hiii Jurgen, 

As per your suggestion, I tried my antiserum using positive and negative control. For negative control I used normal cell lysate and as positive control intact virus. As per standardized conditions, I used 1:75000 dil. Sec. Ab and 1:1000 dil. Pri. Ab. I diluted cell lysate as 1:100, 1:200, 1;500, 1:1000 and 1:2000 in PBS. After dot blot of these samples 1:2000 diluted cell lysate is not showing any signal. But problem is at this dilution of pri.Ab, positive control is not showing any signal. So for positive control confirmation I reduced the dilution of pri. Ab to 1:500 at which I am getting the signal for positive control (Virus). So what is the solution for this situation?

Before this I did one experiment in which I tried to develop a  dot blot of  crude cell lysate  with sec. Ab (without treating the membrane with Pri. Ab). Unfortunately in this case the samples were showing signal. Is there any problem with secondary antibody or ECL reagent?

Can anybody help me out to solve this trouble?

Hi Asia, any endogenous peroxidases will show a signal with the ECL reagent.

(There are a lot of endogenous peroxidases in all tissues).

The best way to eliminate endogenous peroxidases is to boil the samples with SDS and azide before doing the dot blot.  

It seems to me that 1:75000 dilution for Secondary antibody is quite optimistic. These commercial reagents are generally quite good, but we usually dilute them no more than 1:15000. Of course also the ECL reagent can be limiting, the peroxide is usually the breakingpoint. We make our ECL reagent ourselves and we buy fresh peroxide every 12 months. This is easily checked, if other people in the lab use a different primary AB but use the same secondary without problems, then that's a positive control for both ECL reagent and the secondary antibody. If you are the only one in your lab doing immunodetection, and the ECL reagent is old...then you should check the reagents first with a working set of primary antibodies and good antigens.