I want to extract proteins from serum in order to use them in Western Blot analysis but I can't find any protocol for protein extraction from serum. In fact, I would also like to know whether it is possible to do a western blot on serum protein even using the serum itself.
Can anybody help me?
Thanks!
The problem with using serum for a Western blot is that the composition of serum is mostly one protein - serum albumin. Much of the remaining protein is immunoglobulin. Since there is a limit to how much protein you can run in a lane of a gel, the sensitivity of detecting minor serum proteins by Western blot is limited by the presence of so much albumin and immunoglobulin. Fortunately, you can purchase resins that will remove the albumin and, if desired, also the immunoglobulin, greatly improving the performance of Western blots of serum proteins.
It is generally not recommended to use serum directly for Western Blot as it contains many other proteins and components that can interfere with the detection of the target protein. Therefore, it is important to extract and purify the target protein from the serum using a precipitation method i.e add a precipitating agent such as trichloroacetic acid (TCA) or acetone to the serum sample to precipitate the proteins. Incubate the sample at 4°C for at least 30 minutes to allow the proteins to precipitate.
The most critical question is how to pre-treat the serum:
General treatment: conventional separation of serum, that is, after taking blood, it is placed at room temperature to solidify, and then centrifuged at 3000 rpm for 15 min, and the supernatant can be taken.
If the target protein is very low, or if the target protein has a similar molecular weight to albumin or globulin, serum albumin and/or globulin must be removed. Such as Qproteome Albumin/IgG Depletion Kit, ProteoExtract™ Albumin Removal Kit.
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