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8M Urea just lysis the cells, then with SDS samples loading buffer with DTT to the gel. I agree that I used too much of the heart mito lysate.
Asked on Oct 18 2012
Oh, I see.
The bovine heart mito lysate was prepared with iso-osmotic buffer, thus without detergent - so that it can be used for various experiments not just WB.
Additional information as per datasheet:
2 mg of bovine heart mitochondria in iso-osmotic buffer. This sample can act as a positive control in immunocapture applications and Blue Native PAGE.
1 mg solubilized mitochondria for Immunocapture experiments. For the Immunocapture of OXPHOS complexes, it is recommended that 180 µl of the mitochondrial membranes supplied be solubilized by the addition of 20 µl of 10% lauryl maltoside. Insoluble material is removed by centrifugation at 72,000g before immunocapture of complexes from the supernatant. For more detail see Abcam's suggested protocols.
Thus, the preparation of the lysate in this way, works to obtain the expected band at 37 kDa. It seems therefore that the use of 8M urea has a different affect on your samples which might cause it to run at the higher MW.
I would suggest - in light of your results - to try lysing your cells either with an iso-osmotic buffer (mechanical lysis) or an SDS-based buffer. That should help with obtaining the band at the expected MW.
I hope this is of help. Please let me know if you have more questions.
Answered on Oct 18 2012