I am performing some WBs to detect MMP-10 in mouse skeletal muscle tissue. MMP-10 as zymogen is ~54KDa and when active is ~45KDa, but all my samples show one single clear band of ~85KDa. The positive control is purified rhMMP10 and does appear at its normal weight (45KDa).
I've tried different homogenizing protocols (with phosphate inhibitors, with proteases inhibitors, with polytron homogenizer alone, and also followed by homogenizing through decreasing diameter needles) but I get the same result every time. For denaturalization I use the invitrogen reducing agent and 10 min icubation at 60ºC. I have also tried at 95ºC but still no changes.
It looks like MMP-10 is tightly bound to something in the tissue that is very difficult to separate. Does anyone know what it could be?
Thanks!
It seems to me that maybe the antibody reacts with something else. Under denaturing conditions, the things that might bind MMp10, like TIMPS would be dissociated. An alternative is that because the MW of the monomer is 45 Kda, and the band you see is about 90Kda, that it is a dimer, and you may not be adding enough reducing agent to your cell lysates/loading dye.
Can you just add a large excess of fresh DTT to the loading dye and then heat it. I am not sure about the preparations you get from companies. Typically, one needs fresh reducing agent unless it is TCEP.
I agree with Marcia. Also to me it seems that there might be not enough DTT for a complete reduction. Or it is a simple (and unfortunately frequent) cross reactivity with something.
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