Hi everyone,
I have some important problems with my western blot against serum proteins. I have used secondary antibody conjugated by HRP that binds in unspecific manner to other proteins as same position as my desired protein, like in 130, 180, 50, and 25 kD molecular weights. I have done over western blot without using primary antibody but using secondary antibody, and see these results.
What should I do that my secondary antibody don't bind to other proteins in PVDF?
Hello Fatemeh Vahdani
The serum contains many proteins that could result in non-specific binding. Albumin and IgGs are the most abundant proteins which tend to obscure the signals of less abundant proteins, preventing accurate detection. By depleting samples of albumin and IgGs, the quality and depth of the analysis can be greatly enhanced. Depletion of the two would remove more than 60% of the total protein content in your sample, allowing proteins normally obscured by albumin and IgGs to be visualized. So, you will have to optimize your serum sample preparation by removing albumin and IgGs using depletion kits which are commercially available so that excess of high-abundance serum proteins are removed and less-abundant proteins of interest may be detected.
After having carried out the clean-up procedure, I suggest you perform the below steps for better results.
a. Load a lesser amount of serum sample since it may reduce background to a certain extent.
b. Use Nitrocellulose membrane instead of PVDF as Nitrocellulose membranes are generally thought to give less background than PVDF.
c. Preferably carry out an overnight blocking.
d. Use a higher percentage of Tween 20 in the wash buffer and increase the duration of washing steps.
Hope these suggestions help!
Best.
Thanks dear Malcolm for your answering,
My goal to do western blot of some specific protein in serum is following of them to recognize upward and downward of them during progression of disease. Do you think this kit can useful for my goal to show difference in these proteins on western blot and has no effect on levels of my proteins?
If those proteins do not stick to albumin it will not be a problem. Type the Human Albuminome to Google to learn much about the sponge effect. The depletion process may lead some markers to disappear undesirably. If your targets are not one of them do not hesitate to remove IgG along with albumin to decrease non-specific binding and to report up or down-regulation of your proteins. (Do not forget to include Housekeeping proteins or total protein normalization to verify the assay)
Good luck
İsmail Emir Akyildiz Malcolm Nobre can you tell me a protocol to deplete of immunoglobulins of serum except kits or immunochromatography?
Procure a protein A or G ligand attached resin and apply it to plasma. You do not need chromatography. Either batch mode, gravity flow, magnetic bead assisted, or spin column format can bind IgGs and wash out your targets. Immunodepletion would be the most effective approach and can be performed w/o chromatography. Conventional chemical precipitation or physical isolation methods will cause your proteins to be lost. Depending on your choice of analytical platform, protocols can be further discussed.
Thanks Ismail for your answering.
I have done IgG depletion from serum, then western blot with reducing and non-reducing SDS-PAGE, in both I had non-specific bands in 25,50,130 and 180 KD.
Now, I don't know what should I do on detecting a specific protein in serum.
On the other hand, I know specific secondary antibody against intact Fc, can be useful based on articles, but I am not sure.
Do you have any idea that can be helpful?
I suggest using both immunodepletion of IgG and albumin removal. Removing only IgGs would not be sufficient. I indicated protein A&G to remove IgGs due to the question you asked based on a protocol to deplete immunoglobulins of serum except kits or immunochromatography...It is always better to remove albumin first.
Secondly, a better selection of primary antibodies and aggressive washing and blocking steps are needed along with extended incubation of probing at well-established Western protocol.
Specific secondary antibodies against intact Fc conjugated with HRP are useful agents to monitor your specific protein if they are combined with primary antibodies. Be careful also about species compatibility at the western setup for secondary against primary selection. Do not skip to use of primary antibodies to enhance selectivity.
Your target protein's specific AA sequence should be the antigen for the paratope of your fishing out antibody not the every Fc portion of each antibody in sera. Therefore, you need to apply primary incubation followed by secondary probing.
Best
Thanks İsmail Emir Akyildiz ,
About appropriate binding of primary antibody and specific antigen in serum, I'll do immonuprecipitation to catch specific antigen then check it with primery antibody. But about WB for whole serum,
What is your idea about depletion albumin without kit. Have you ever done it without any kit?
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