Using the DuoSet Human TGF-B1 ELISA Kit of R&D Systems (#DY240-05), which detects active TGF-B1, the protocol suggests you to activate the latent TGF-B1 of the cell culture supernatant by adding 20uL of 1N HCl into each 100uL sample for 10 minutes at room temperature. This step is followed by the addition of 20uL of 1.2N NaOH/0.5 HEPES into the sample, to neutralise it. We measured the pH of both the HCl and the NaOH, as the protocol suggests, but when comparing the samples that were not activated with the samples that we activated, the not activated samples show a greater OD, suggesting that there is more activated TGF-B1 when we do not activate it using the sample protocol.
Any suggestions?
Hi Silvia, I have used the DuoSet you describe in the past. If acid activation is problematic you can heat activate latent TFGb in your conditioned media by heating at 85 Celsius for 5 mins. This has worked for me in the past in both ELISA and TGFb reporter cell systems. I'm not sure why your signal is lower in the non acid activated sample - maybe you are denaturing the mature peptide? (could you tirate the HCL concentration down until you find the effective concentration?) For example, if you heat L-TGFb above 90 celsius then it will also begin to denature the mature peptide.
I would just switch to heat activation!
Hope some of that helps....
Hello Robert, thank you for your answer!
We also thought one possible explanation was that the mature peptide was being denaturated in the process, but we were not sure, as many people used the same kit following the same activation protocol and seemed to not have that problem. Your suggestion was very useful, we will try the heat activation next!
Thank you so much,
Sílvia
Hello Robert, thanks for your suggestion!
I currently also have some problems in measuring TGFb in my conditioned media. I would like to try your heat activation next time! Can you give me some advice on how to deal with the frozen sample? Shall I let them thaw in room temperature and then 85 Celsius for 5 mins in the waterbath?
Thank you so much,
Luxi
Luxi Ye. I usually heat activate media directly after cell culture and proceed directly to ELISA/reporter assays.
However on a couple of occasions I have used frozen conditioned media. In this case I thawed at RT and heat activated as you described. In my expereince this worked well, but I cannot say I optimized the procedure. I also did not directly compare the signal differences between fresh and frozen.
I should add that whilst the TGFb duoset appears to have limited cross-reactivity with Bovine TGFb, my reporter cells respond well to bovine mature TGFb - so I found it to be much cleaner to use a serum free culture protocol if that's possible for your cells.
Sorry I can't give more help.
Rob
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