Its difficult to suggest on the basis of your little information provided. It seems that you are getting high background on WB using serum sample.

As you know, the serum contains number of proteins including antibodies that can increase NSB and give high background on WB. The blocking buffer itself has several limitations. For instance, animal serum containing un-purified primary Ab can bind to animal proteins in blocking reagents. In addition, non-fat dry milk can specifically react with certain antibodies or detection reagents. Milk contains casein, a phosphoprotein that can react with phospho-specific antibodies. Milk also contains variable amounts of avidin that can interfere with avidin-biotin detection systems. Both of these interactions will result in high uniform background.

There are several factors that can generate high background. Few major are the type of membrane, age of membrane, washing of membrane, overexposure of membrane etc.

If you are getting problems only with serum sample but not in other samples, then try to set your goal for which you are running WB. You have to optimize the immunodetection system in you lab in order to get specific signal by keeping in mind the all possibilities that generate high background with serum sample (as I have described above).

Moreover, if you think that your protein of interest is very low in amount, you can go for immuno-precipitation using your antibody and then eluting and running the gel.

Since serum contain large amount of albumin, you can remove the albumin using resins from serum sample or deplete the serum sample. For this purpose, you can use IgGs and Albumin removing kit from minipore (122642 | ProteoExtract® Albumin/IgG Removal Kit) in order to reduce the albumin and IgG from serum sample. Hope this may help to overcome your problem that ultimately could increase the specificity of your WB.

Good luck!

Thank you for your input. The method we are using is not traditional WB but Capillary western/Simple western (Wes system) by Proteinsimple. We have tried depleting albumin and IgG using several different methods, but I think our capillaries still cannot handle the amount of IgG and albumin that we cannot get rid or through our deplesions. We are not having the same problem with the same antibodies using traditional WB.

Best wishes

Anna

Dear Anna Kendall

If your protein of interest is smaller than 30 kDa, you can use respective protein concentrators/centrifugal filters and use the filtrate for the analysis.

Thanks, interesting suggestion. I will definitely look into it! Our protein of interest is 27 kDa. Do you have personal experience with this method? Any particular brand that is better?

Best wishes

Anna

I used Amicon® Ultra-4 Centrifugal Filter Units and Amicon® Ultra-15 Centrifugal Filter Units (available from Sigma Aldrich/Merck) 30,000 NMWL for serum and they worked very well. After this I used also sequentially 10,000 NMWL and 3,000 NMWL. In that way I obtained several fractions of serum with different molecules in them. I used this fractionation for different application than yours, but I think it can work for you as well.

Best wishes

Tomasz