I would like to measure BDNF in muscle. I am currently using a commercial (pre-coated) sandwich ELISA kit, however I seem to be running into issues that I believe to be the result of muscle having low BDNF levels.
1) Serially dilution of samples does not result in a corresponding change in OD/concentration for MUSCLE. However, spinal cord samples and BDNF standards look fine.
2) I am working with a limited dilution range (for this reason, my serial dilution consists of only 2-3 samples). This is because muscle contains low amounts of BDNF and so I can't dilute very much. On the other hand, I must dilute otherwise I will have interference for antibody binding (due to components of the homogenizing buffer- NaCl and Triton).
3) If there is nonspecific binding occurring, is it possible to purify BDNF from my samples? I have heard this protein can be unstable.
Do you have any ideas/suggestions of why I may not be seeing a difference in my serial dilutions? Are there other ways of measuring BDNF at such low concentrations?
Your help is very appreciated. Please let me know if I can provide any clarifying information!