Hi,
I'm currently trying to blot for VCAM-1 produced by endothelial cells in response to TNFa stimulation. The issue I'm having is that I am yet to see any bands for VCAM-1!
I have tried three different endothelial cell lines, two primary (HDMEC, HUtMec) and now an immortalised cell line HMEC-1. I have tried incubations with TNFa at concentrations up to 40ng/mL and durations of up to 24hrs. I have tried NP-40, RIPA and Co-IP lysis buffers and am achieving 1-3mg/mL of protein in the lysates. My antibodies (VCAM-1 from R&D and Licor secondary) are working fine, as I have run a positive control to check for this being the issue.
So, do any of you have any suggestions? I have looked through the literature and can see that this has been done before, and even following these protocols have not yielded results.
Thanks in advance!
Try with a high sensitive chemiluminescent solution like them from Advansta (https://advansta.com/products/western-blot-substrate-WesternBright-ECL/). You can detect here the bands of your interest protein even if they are very very low.
Second recomm., would be to transfer to PVDF memmbrane with longer time periods (instead of 1h you should try it with 3 h).
Good luck!
Hi Jack,
Please check out this paper (sorry for the self promoting), we did some similar experiments using TNF-a and we can detect VCAM-1 upregulation in human EC by both gene expression and also western:
Paneghetti and Ng Vascular Cell (2016) 8:2
DOI 10.1186/s13221-016-0036-4
Good luck with your study.
Hi there,
I did some experiments last year that were pretty much identical to yours and didn't have any problems getting VCAM-1 bands, the major difference being that I stimulated the cells with IL-1B rather than TNFa - I used concentrations up to 10ng/mL. Maybe you could try stimulating your cells with something else and see if your TNFa is the problem?
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