we usually use RT-PCR to determine BDNF gene expression and Western blotting to measure BDNF protein in rodent brain. If we decided to use gel electrophoresis instead, would it be more accurate to measure BDNF gene or protein levels?
Furthermore, is there any other technique to assess BDNF levels in rodent brain?
Hello
Look at these links
Good Luck
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4675070/
http://www.merckmillipore.com/INTL/en/product/ChemiKine-Brain-Derived-Neurotrophic-Factor%2C-Sandwich-ELISA,MM_NF-CYT306?ReferrerURL=https%3A%2F%2Fwww.google.com.sa%2F&bd=1
To detect BNDF in murine brain samples the first choice would be to perform a western blot on tissues extracted with a relatively light-detergent buffer (e.g. RIPA with 0.1% SDS). Take car with this however as your antibody may well recognize two primary BDNF bands if it cannot distinguish between the mature cleaved form or the pro-BDNF form. Also, direct measurement of BDNF can be made using an ELISA assay - we used to use Promega but then this assay was eventually withdrawn - other kits are now available from R&D Systems as well as Abcam. I hope this helps ! - Cheers, Stuart
We agree with Stuart about the presence of at least 2 forms of BDNF, the mature and pro forms. The many different antibodies that are available will localise one, or the other or both. A truncated form of BDNF has also been identified and quantified by WB in blood (see publications by Tongiorgi's group). There is no evidence that this form is in brain extracts, but you need to watch for it at 28kD in reduced gels.
The publication identified by Saleh above (Polacchini et al, 2015) is a really important analysis of 6 different commercial BDNF ELISA assays. Biosensis was shown to have the fastest, most accurate, reliable, and most importantly the best batch to batch reproducibility. Biosensis has worked with leading researchers to achieve this quality assay. We suggest you use this assay in tandem with the specific proBDNF ELISA kit to independently quantify both the mature and pro isoforms. However, please be aware that I declare a financial interest as a shareholder in Biosensis.
You can talk to the scientists who developed the ELISAs at "[email protected]"
I greatly appreciate your answers and sharing your experience. I will put your advices in my consideration. I can conclude from all your answers is the best way to quantify BDNF is the ELISA then western blotting. Electrophoresis is only for detection BDNF.
Dear Ali,
Thank you for you comprehensive answer. I got what you mean.
Has anybody observed 20-22 kdA BDNF band along with 14kda mBDNF, 28 kDA truncated BDNF and 32 kDA Pro-BDNF? What is this 20-22 kDA BDNF band?
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