1 reply to this topic

[jaxrich]

Hi there,

I have been working with mouse tissue for quite some time. It has recently come to my attention that the goat anti-mouse antibody (GE) I have been using causes very crisp, strong bands at 50 and 25 kDa in the absence of primary antibody. This also happens, though not nearly as strongly (about 1/10th as strong) in rat tissue. I also saw this using a donkey anti-mouse (Jackson), only much stronger and even more bands. I have not had a chance yet to try no primary with these samples plus a rabbit secondary, but I suspect the bands would not show up, based on some other blots where I used a rabbit secondary and there are no bands at 50 kda.

50 and 25 seem like heavy and light chain fragments, but one professor I talked to said if it really was endogenous antibodies it would look like a big smear and not sharp bands. That is about all I have right now.

Any input would be greatly appreciated.

[CellSpecific.com]

I assume you ran these under reducing conditions. Have you ran these under non-reducing conditions? If these are antibodies, under non-reducing conditions you should no longer see the 50 and 25 KDa bands but rather a single ~150 KDa band which would further support your suggestion that these are antibodies. Let us know.