I have two unique teleost species, Saffron Cod and Broad Whitefish. I am analyzing the HSP70 concentrations in the cranial, hepatic, and muscle tissues. The Broad Whitefish samples all worked perfectly. However, the liver and muscle tissue samples for the Saffron Cod aren’t running (the cranial tissue samples are working perfectly). The HSP70 protein seems to be aggregating and sitting on top of the well.

My advisor and I believe perhaps the cod have a higher concentration of fats and thus aren’t separating like the other samples. All of the tissue samples were prepared the same way using a homogenization buffer with 4% SDS and Tris-HCl. The tissue samples were homogenized in the buffer, boiled for 5 minutes and spun down at max RPM for 15 minutes before extracting the supernatant with the protein sample. The protein samples are placed in 2xSB and boiled for 3 minutes before being loaded in 10% gel. All of the solutions, both cod and whitefish, are clear and fluid when at room temperature (none are gelatinous or are behaving weirdly). I have attached images of the gel and Western Blot with four Broad Whitefish samples (BDWF) and four Saffron Cod samples (SFCD). Muscle tissue is MT, hepatic tissue is HT, and cranial tissue is CT. The Western Blot for the Saffron Cod muscle sample didn’t show up for some reason, but when I ran it the other day it looked exactly like the liver tissue; the band was sitting at the very top of the well. Thank you for any and all help!