Hi everyone,

I'm getting a band (probably unspecific binding), under my housekeeping COXIV protein. I think it's probably secondary antibody cross reaction, but I'm not entirely sure.

On the first picture, the upper two bands (around 60 kDa) show the protein of interest, double bands represent the two different protein isoforms. The lower bands (around 15 kDa) show bands of the COXIV housekeeping protein. Tissue is adult mouse brain, amount of protein loaded 10ug, RIPA buffer lysate, Laemmli boiled samples.

As you can see, I've cut the membrane and incubated primary and secondary antibodies separately and the results are great.

-Primary ab: a-rabbit CTR 1:1000 in TBST, a-mouse COXIV 1:500 in 5% milk

-Secondary: ScanLater a-rabbit and a-mouse goat (EU-labeled for use with SpectraMax), both in 1:5000 TBST, incubated seperately for respective primary.

Second picture shows what happens if I incubate both primary and secondary abs together. I get this second band above the COXIV around 20 kDa and the intensity doesn't change with the amount of protein loaded. I'm not newbie with WB, so I think washing steps and buffers are well controlled. I think it might be some kind of cross reaction of secondary abs, since these new ScanLater, I've only started using recently. Weird thing is that some people in the lab in the past, have followed the exact same protocol and they were getting one band.

Any contribution would be highly appreciated, can't get my head around it it seems.

Thank you in advance!