I am looking for a ELISA protocol to analyse TGF-Beta 1 using rat hippocampal homogenate. Can anyone suggest, which species antibody should I use for it to get good result (I will be using rat hippocampal homogenate to analyse TGF-Beta 1) it would helpful if anyone could provide the protocol or articles (since many researchers used western Blot).
We've been using 'instant' kit by R&D for mouse TGFb which is also validated for rat. For procedure - follow the datasheet. It's good to check also latent TGFb level ;)
http://www.rndsystems.com/Products/MB100B
We at our lab use BD Biosciences kit for quantification of TGF-beta. It works quite well. The other options include kit from e-biosciences. The procedure is quite simple. Just coat primary antibody in 96 well plate supplied with the kit in the coating buffer overnight at 4 degree, followed by 3 washes with PBSt. Then add standard and samples to the wells, incubate for an hour at 37 degree and wash with PBST. Now add HRP conjugated secondary antibody, incubate for an hour at 37 degree, followed by washings. Finally add substrate and wait for 10-15 minutes for color to develop. Stop the reaction with 2N H2SO4 and read plate using a microplate reader.
We used to quantify rat TGF-beta1 with the Human DuoSet kit from R&D System. It works quite well, is cheep and easy to use.
For note, we also try a kit from Promega, but it failed to detect our sample.
I am also interested in assaying for active TGFB1 in whole brain homogenates from mice. I have two questions, and would appreciate your help:
1) How did you homogenize your brain tissue for the TGFB1 ELISA? If you have a buffer recipe you found appropriate, I would really value if you shared it with me.
2)When using commercial ELISA kits for TGFB1, such as the kits from R&D that are not typically validated for tissue homogenates, did you follow the sample activation steps for cell culture supernatant or the steps for plasma/serum? Similarly, what samples diluent did you end up using? As this kit has two sample diluent buffers, one if using cell culture supernatants and one if using plasma/serum samples, none if using tissue homogenates.
Manual for R&D kit:https://resources.rndsystems.com/pdfs/datasheets/mb100b.pdf
Thanks,
Rania
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