I am trying to quantify a peptide in soybean leaves using ELISA but I couldn't. I think I have some interferents from the extraction buffer, then I dialyzed the sample before adding in the ELISA plate and I did not have success also, the readings are similar to the blank samples. I believe is a problem in the assay because I used the same sample to perform a Western Blot and the band appeared. Someone had the same problem using ELISA to quantify plant proteins? I am using alkaline phosphatase. Thanks!
Hi Fernanda, are you looking for soybean proteinase inhibitors?
No. It is a transgenic soybean. This plant express an insecticidal peptide and I am trying to quantify.
Dear Fernanda,
I think after extraction, you should try to concentrate the sample before the ELISA. But then, what did you dialyzed against..the same extraction buffer?
Hi Fernanda, RIPA buffers or something similar can interfere with the ELISA. Dialysis will not help if this is a detergent problem.
Hi Dear Ayodeji,
We dyalyzed against Tris-HCl 50 mM and NaCl 0,15 M, pH 7.4. This is the buffer that we use to perform ELISA. We are using ammonium acetate in methanol and acetone to concentrate. Today, I changed the concentration protocol and I am using 100 % TCA and acetone. I will see if I will have the same results.
Thanks for your attention,
Fernanda
Hi Dear Stefansson,
Thanks for your help. Maybe I need to remove the detergent to avoid this problems with ELISA. I will try this approach.
Best Regads,
Fernanda
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