Is myc expressed in hippocampal neurons? - endogenous myc expression in hippocampal neurons? (Jun/28/2006 )

I've started in a new lab recently and inherited pcDNA3.1 myc/his tagged constructs. I transfected these into primary rat hippocampal neurons and ofcourse did a negative and empty vector control. When I do immuno with a monoclonal myc antibody (clone 9B11 EQKLISEEDL) I got positive staining in my empty vector control. I checked whether the vector is in frame without an insert and makes myc, it does not. I then just took some hippocampal neurons and performed myc immuno on them and got positive staining. Looks like i've stained with tuj (ie..its in the cell body and axon). I've searched the web and pubmed and many people use myc tagged vectors to transfect hippocampal neurons. I even checked the epiptote in methods sections in some of these papers and it is the same as mine.I have come across a couple of refs that show that myc is expressed in the hippocampus. The only thing I think of is that the particular clone i'm using is giving me this result or I've somehow contiminated my antibody. Which I have test by borrowing an aliqout and testing this on these same neurons. If any one has any clue as to what is going on, feel free to post a reply. Any help would be greatly appreciated.


Jay

I have used myc antibody and had problems with retinal staining. Although didnt have much of a problem with cells in culture.

Found this, may b its helpful

"c-Myc is a protein crucial for cell cycle progression but also is up-regulated in several paradigms of neuronal degeneration (Qin et al., 1999; Di Giovanni et al., 2003)"

A quick PubMed search shows that c-myc expression is inducible within hippo neurons, and should be low in the 'normal' cell. It is interesting that you're seeing it throughout the cytoplasm and not within the nucleus though. Please post your results using a different antibody.

Can we have a look at the 3 pictures, please? No need to say which is the protein, just call them no vector, empty vector, myc-protein or something like that. Hope you will show us, because this can really help.
Moreover, you could check your antibody reactivity. The Abcam clone 9E10 doesn't react with rat, while they don't specify for the 9E11. If you buy one that doesn't react with rat, you should solve the problem

Thanks everyone for their reply and interest in my question. I'm new to this website, so I dont know how to post the pictures. I will post all pics tomorrow (once i figure out how) and after I finish my experiment with the 9E10 clone that I borrowed from a friend.

Thanks again

Jay