Seems like problem with the usage of too much primary antibody. Try after reducing the concentration of primary antibody. Another possibility is insufficient blocking and membrane drying.

There are several factors responsible for nonspecific binding. Ideally one would heretically standardize the conditions as per the antibody dilutions recommended by supplier but apart from that one should look into the blocking reagent ( Non fat milk is preferred but in my case 3% BSA for 2hours works better). You may want to increase the time of blocking - no washing - decrease the primary antibody incubation time- washing- decrease the secondary antibody incubation time- washing. You may also try putting 0.05% tween in primary and secondary diluents. To me it looks like a secondary antibody nonspecificity

All the best.

I am not sure if I am missing something so first let me ask you for a clarification... what are you using as your primary antibody and what are you using as your secondary antibody?

Hi

thanks all of you for the answers!this is what I normally do:1-after transferring I incubate the membrane for 1 hour with super blocking solution or 5% milk blocking solution (both with 0.05% tween) 2-incubate with primary Ab overnight (in this membrane I used Beta actin diluted 1:1000,Ab75186) 3-washing 3x PBS 4-incubate with secondary Ab (anti rabbit,Ab16284) for 1 hour 4- washing 3x PBS 5- Developing using the ECL technique (exposure in 11.1 sec)

do you think I should incubate with blocking solution over night?

Elahea, incubation in primary antibody with blocking solution overnight can be a good option to reduce non-specificity. Also you try to reduce the primary antibody dilution to 1:1250 or 1:1500.

OK, it's human plasma and you use secondary a-rabbit. Did you run a control with no primary, to see the cross reactivity of your secondary with the human antibodies in your plasma? Perhaps you can do dilutions of your secondary and do a mock primary incubation (keep the same time in whatever you use to dilute your primary, but don't add it). Do you see bands there? Does it get better with a more diluted secondary? Let us know how it goes!

thanks Sivadas and Claudia for your answers. yea,I tried the cross re activity of the secondary with antibodies in the plasma with no primary and there was no binding! so I think there should be an other cause for these unspecific bands. But I will try mock primary incubation and let you know :)

May I ask what are the corresponding sizes of the major bands that appeared on your blot?

I tried mocking with primary Claudia!also decreasing the dillution of primary Ab but in both situations there was no band!

the corresponding sizes of the major bands that appeared, the darkest band is 25 kDa where as I used Beta actin antibody on this membrane (42 kDa)

I've been running human plasma samples as well, if u're running at a reducing condition, it is likely that the observed band at 25kDa is igg light chain, chances are the other bands u see be ~55kDa and 100kDa, corresponding to igg heavy chain and heavy chain dimers respectively. Semi reducing condition would likely reveal a ~130kDa igg. What are the sizes of the top 2 bands in the picture?

ok that makes sense, two top bands are 90 and 140 so yes they are probably IgG but is there any way to get rid of them or prevent antibodies from binding to IgG?

I do hear of the use of protein A or protein G conjugated with HRP as they only recognize folded Ab, lesser extent to unfolded ones. But by the sounds of what you had mentioned earlier, it seems like the primary Ab is the offending Ab. I'd think the next step might be to remove albumin and igg by commercial kits. I'm not too sure of other methods, sorry about that.

Elaheh, please read this article, "β-Actin as a loading control for plasma-based Western blot analysis of major depressive disorder patients" (http://www.ncbi.nlm.nih.gov/pubmed/22617797).

Hope it can help you.

I read that before,anyway thanks for the respond