We want to do western blot in human serum in order to validate some proteins with changes resulting from iTRAQ-MS/MS data. Is it possible to do western blot directly in human serum without problems? In the case that the answer is yes, should we charge the gel by volume or by total protein amount?
You mean using the serum as sample? Sure, it's done all the time. The only problem is that serum contains some proteins in high concentration (most notably albumin and immunoglobulin, together they can account for about 90% of total protein), others in very small concentrations. The total amount of protein in SDS-PAGE is limited, so there may be to little of your protein in that amount to be detected.
There are affinity matrices for albumin and IgG removal (for example doi:10.1016/0009-8981(73)90341-0, doi:10.1002/pmic.200402048, doi:10.1002/pmic.200300465), but they may also remove other proteins.
If you have determined the protein concentration in your serum samples, you can use the same volume of serum for electrophoresis, it would be easy to convert signal per µL into signal per mg, should that be desired.
You mean using the serum as sample? Sure, it's done all the time. The only problem is that serum contains some proteins in high concentration (most notably albumin and immunoglobulin, together they can account for about 90% of total protein), others in very small concentrations. The total amount of protein in SDS-PAGE is limited, so there may be to little of your protein in that amount to be detected.
There are affinity matrices for albumin and IgG removal (for example doi:10.1016/0009-8981(73)90341-0, doi:10.1002/pmic.200402048, doi:10.1002/pmic.200300465), but they may also remove other proteins.
If you have determined the protein concentration in your serum samples, you can use the same volume of serum for electrophoresis, it would be easy to convert signal per µL into signal per mg, should that be desired.
Thank you very much,
indeed, I was wondering about the fact that 90% of protein in serum is Albumin and IgG and I thought that it could be some interference as unspecificity due to that issue.
I agree, this is possible. You need an adequate sample environment (e.g. detergences, pH, conc of isons etc.) and should charge gels with comparaple protein conc.
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