Verify the protein spots found by 2D using Western blot - The expression level change are different (Aug/24/2006 )
I am studying on what protein lelvel will change when the drug are treated to certain cell line.
By 2D and MALDI-TOF-TOF, I found several protein level change, so I choose some interesting protein to be verified by 1D western blot.
Some protein level change are the same in 1D and 2D,
but some can not be verified, meanes the protein level change are contrary in1d and 2d !
Can anyone give me an explnation, how can I interpret this result?
Should I change different antibody and try?
Thanks a lot.
The changes in protien level which u observed in 2D, was it the result of one gel or a few 2D gels?
I think that there is no need to present both 1D and 2D gel, so 2D separation is the better choice to demonstrate proteins and immunostain; in 1D, a protein band may represent various isoforms of same size which are separated by 2D and "streching" the immunosignal to lesser local intensity than on 1D blot; when using ECL-films the slope of sensitivity is relatively flat and needs a threshold which means "strechted" to weak signals on 2D blots may not be detected but if condensed in a single band on 1D are added to an alleged stronger signal; better use an imager