I have isolated serum from rats and I want to detect growth factors in the serum. I want to know how would I have to prepare my serum samples and which dilution range should I use for my serum?
When analyzing proteins present in serum in low concentration I suggest you remove albumin and immunoglobulins prior to westen-blotting. They account for >60% of all the proteins in serum and could potentially decrease the sensitivity of your blotting experiments. There are commercial kits available that you can use!
Good luck with your experiments!
Christian
Thank you for the suggestion but the problem is I don't have the kit for albumin depletion and I have limited time to complete the experiment.....how should I go about without albumin and IG removal?
Hi Maria,
there is a limit on how much protein you can load onto a gel (depending on gel size, well dimension etc.) - loading too much will lead to migration artefacts. Another problem with albumin and immunoglobulins might be that these proteins will saturate the area of the blotting membrane masking / inhibiting the immobilization of proteins of interest in the corresponding molecular weight region (albumin 60-70kDa, immunoglobulin chains heavy ~50kDa, light ~25kDa).
A crude procedure to remove immunoglobulins might be to use protein-G sepharose (mix with serum, centrifuge and load the supernatant onto the gel). However, this leaves you with albumin, but maybe your growth factors differ sufficiently in size to that it won't bother you?
Also bear in mind that - if you use detection via secondary antibodies (e.g. anti-IgG) - this will give you a very strong background signal (especially if your first antibody is from mouse). You should check cross-reactivity with rat immunoglobulins of these secondary antibodies first before you start with your growth-factor experiments!
I hope that helps a bit!
What should be the centrifuge speed if I use protein G sepharose. Should I use different dilutions of my serum sample to optimize the protocol I was thinking of 1:100, 1:500 and 1:1000 or should I load 50ug of total serum protein?
After Albumin depletion would I be able to proceed my serum sample as any other protein sample for SDS PAGE. I have read it requires desalting after the albumin removal?
Hi Maria,
it should be fine to simply dilute your serum samples, add a constant amount of protein G sepharose. mix well, incubate and centrifuge afterwards at sufficiently high speed that allows you to remove a clear supernatant for gel analysis. Don't forget your negative/positive controls!
Best regards, Christian
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