I am panning to maintain CD4 Cells in culture for 10 days using T cell activation/expansion kit from Miltenyi to test the effect of a treatment on some proteins by Western blotting, so I am worried that beads (although they will be removed before hand) would affect the protein expression or its detection by western blotting!! any experience or advice regarding that ll be greatly appreciated. Thx
Hi Fatma,
In general, I don't think you should have a problem. We usually remove the activating beads from CD4+ T cells after pre-expanson (about 5 days), and then re-activate in-vitro utilizing soluble anti-CD3/CD4. However, we make our own CD3/CD28 magnetic beads. In our hands, upon stimulation of pre-activated CD4+ T cells, proteins that are associated with the TCR are normally phosphorylated and show up well in the immunoblott (Zeta chain, ZAP-70, LCK, etc...). As far as protein expressing going up/down upon stimulation, it would depend on the type of protein you are looking at. For example NFkB tend to be increased in the nucleus of pre-activated cells compared to un-expanded cells; which is expected. It is also known that T cell down/up-regulate proteins associated with the TCR/LAT upon stimulation such as SLP-76. Overall, I don't think you should have a problem but you should probe the expression of your protein pre and post-activation just to make sure.
Also, If you are worried that left over beads might interfere with protein detection on the immunoblott itself then I would say that as long as you remove most of the beads out you shouldn't have a problem. If you however are stimulating with soluble CD3/CD4 then you will have interference at 25 and 50kD due to the increased antibodies in the solution.
Hope this helps
Mahmood
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