I want to do western blot for cytokines from plasma and serum of rat. Have anybody tried ? I want to know the dilutions and its success rate.
Thanking you.
I think you should provide the exact names of cytokines you wish to analyze. If no literature regarding specific cytokine concentrations in rat plasma/serum is found, you can perform preliminary western blot analysis with different dilutions including all the negative controls - in order to assess an optimal dilution coefficient. Do not forget that inflammation can result in rapid increase in cytokine concentrations.
Thanks Natalia :) well, TNF alpha,IL1,IL6,IFN beta are the cytokines now I am focusing. but all papers sofar I found are with ELISA approach on plasma n serum. If anybody would've tried with western blot,that could be nice information for me :)
Well, possibly, knowing the concentration of specific cytokine in the serum (from the literature, even it was calculated by ELISA method) and based on minimal detection amounts required for western blot (can be found in Ab datasheet) you can "translate" it into western blot. Good luck.
When you say dilutions, are your referring to the primary antibody, protein concentration when loading or something else? Please keep us updated with your progress!
Dear Matt, I'm talking about serum or plasma dilution and loading protein concentration. Do you have any paper(s) regarding cytokines western blot analysis from plasma/serum, Matt? Thanks in advance :)
Sorry, I do not have any papers for this topic but as a rule I have always used a 1:100 dilution for the plasma BCA then loaded 40ug of protein for western blot. If the bands come out too strong use less protein, if they come out too weak increase the amount of protein. Let me know if this helps!
-Also, the data sheet from the manufacture of the antibody should have the dilutions to us for westerns.
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