Human sperm samples from commercial cryobank. Experimental constraints preclude the use of fresh human sperm samples. My preliminary experiments have shown that cryopreserved human sperm cannot be used in WB despite having centrifuged and washed the samples in PBS. This protocol has been successful in the WB of cryopreserved bovine sperm, however there has been no success with human cryopreserved sperm.
did you determine the protein concentration of your samples? Are there any differences in the western blot protocol between fresh and cryopreserved human semen?
Hi Eric,
I agree with Stephanie Thomas. Washing with PBS (perhaps twice or thrice) should actually remove all proteins in the the surrounding medium. Then, check the protein concentration. Finally, for us to answer your question, one would have to know more methodological details of what you did thus far. Which gel staining technique did you use etc. Also, human sperm are much more sensitive to cryopreservation and get much easier damges. Therefore, did you eliminate the DNA for the electrophoresis as this can interfere SDS-PAGE.
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