I’m working on particular species of lizard in India. One of the objectives of my study is to isolate the epididymal luminal protein/s that specifically binds to the sperm surface. In this regard, can anyone suggest an appropriate technique to isolate and identify the epididymal luminal protein/s that specifically binds to the sperm surface?
Additionally, genomic and proteomic sequencing has not been done for this species; but has been done for another species. The quantity of the protein from the single epididymis is usually less, I quantified it by pooling the sample which is approximately
Dear Indumati,
It is not easy to work with a specie where the information about its genome is scarce. I think that you can try first to label your epididymal protein with biotin and then incubated them with your sperm. After washing by centrifugation, you remove the supernatant and you can run a gel with the pellet and reveal the WB with avidin-HRP. The different bands identified will probably correspond with the proteins that you are looking for.
You have to solve the problem of protein identification, I recommend the proteomic approach of the different spot identified.
I hope that helps.
Regards,
Dear Indumati,
It is not easy to work with a specie where the information about its genome is scarce. I think that you can try first to label your epididymal protein with biotin and then incubated them with your sperm. After washing by centrifugation, you remove the supernatant and you can run a gel with the pellet and reveal the WB with avidin-HRP. The different bands identified will probably correspond with the proteins that you are looking for.
You have to solve the problem of protein identification, I recommend the proteomic approach of the different spot identified.
I hope that helps.
Regards,
Manuel Aviles,
Thanks for your suggestion. It seems to be a reliable method which I can adapt.
I'll get back to you If I need any help.
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