We are trying to measure thymosin beta 4 protein expression in HUVEC cell line and in mouse muscle and brown adipose tissue using western blot technique. However, we are not getting any band or signal thymosin beta 4 antibody purchased from Millipore.
Any suggestions?
You need to check the solubility and stability of your proteins in lysis/extraction buffers. Did you check the total cell lysate, soluble fraction and insoluble fractions?
Hi, Is ur protein thymosin beta 4 protein found in SDS-PAGE at the same MW compared to std thymosin beta 4 protein? If yes so u have a problem in antibodies used in Western Blot.
The Antibodies what you have perhaps would have been generated using recombinant thymosin beta 4 protein which would have been conjugated with other proteins and then antibodies would have been raised. But detection in the native system such as cell line/ tissue lysate presents a problem of detection owing to the small size of the thymosin beta 4 protein protein which is 41 amino acids especially in SDS/ western blot. So I think you could go for ELISA or even perhaps 2D gel electrophoresis followed by wetern blot with your cell lysate.
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