We are using BDNF kit to detect BDNF in rat muscle homogenate.
The standard curve is correct, so the kit is working well, however in our samples have a lot of variability in different concentrations. We can not reproduce the technique with the same samples as it once gives us a concentration value X and the next time gives low values, that it seems that we are not detecting BDNF.
I would be helpful if somebody can give me some information, if you know whether BDNF is degraded so quickly that in some cases can not detect it, whether we should add something special to the homogenization buffer or if I have to process the samples specifically… Thank you!
The question is, what kind of kit you use. Try to centrifuge your samples to avoid debris. I guess muscles are quite solid. Sometimes acidifcation may help to release BDNF from membranes. However, absolute value might be still under detection limit. We have done analyes from brain tissue only. It was working.
An ELISA kit from Millipore works very well. Add sufficient amount of protease inhibitor cocktail to inhibit degradation.
Thank you so much for your help! We are working with BDNF Emax® ImmunoAssay System (Promega). We take care about don’t perform freezing and thawing cycles to the sample, to add protease inhibitors (lysis buffer) to prevent degradation, and we tried with different lysis buffer and we also tried homogenizing in different ways... and my absorbance values always stayed around the low end of the standard curve (sometimes falling below)…
Now we are gonna try acidification procedure...will see! also we are considering millipore kit...
Thanks!
Erica,
I'm in a similar boat as you are. I am using the Millipore kit and have had mixed results. I am comparing BDNF levels in various tissues including muscle. I've piloted the kit using brain and kidney lysate and it works but have had difficulties with my muscle samples as well. I received a protocol from another laboratory which also used the Millipore kit that has had success and would happily share it if you would like.
As far as tissue homogenization I have had really good luck grinding the tissue in liquid nitrogen with a motor and pestle. I had used a hand homogenizer with pathetic results earlier.
Hi Mandy,
Thank you for your answer! I would be very grateful if you could share the protocol... mi mail is [email protected]. Also I received some practical tips from other labs that I can share If you would like
And acid treatment also appears to be working…Thank you!
Hi, I have also tried the Emax from Promega accoridng to the kit protocol with the acidification. Values were also at lower end of the standard curve. What worries me most is that we had almost no spike recovery when BDNF was spiked before the extraction. Does anybody have a good protocol?
Hi everyone, although this discussion dates some years ago, I am adding this information which may be helpful to other scientits facing similar problems. We compared the reproducibiity and sensitivity of various commercial ELISA kits and we published the results in:
A method for reproducible measurements of serum BDNF: comparison of the performance of six commercial assays. Polacchini A et al. Sci Rep. (2015)
https://www.nature.com/articles/srep17989.pdf
Please refer to this paper and good luck!
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