I'm measuring protein expression across human embryonic stem (ES) cell (as control), differentiated ES cell and E15 fetal mouse tissue (presumably same stage as differentiated ES cell) by western blot. attempting to detect transcription factor which found to heterodimerize with 90% structurally identical analogue. Result: No band for control (as expected), a band for differentiated cell, and 2 bands of mouse tissue (of different size). What could be reasonable explanation behind different protein size of differentiated ESC and mouse counterparts apart from ab specificity. using polyclonal rabbit IgG.
Why do you think it isn't antibody specificity?
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What about post translational modification/s? Splice variants? Shared epitopes?
Why do you think it isn't antibody specificity?
Send a picture.
What about post translational modification/s? Splice variants? Shared epitopes?
Thanks for the reply. earlier I was thinking that my ab was not specific enough detect the protein. and about to buy new monoclonal but I was trying to find out if theres any other possible modification that could recapitulate different bands (double vs single) observed in differentiated stem cells and mouse tissue. Is that common to see this vriation in stem cells?
First, you need to check the mouse genome databases for alternatively spliced isoforms. These can be found at UCSC genome browser or at the ENSEMBL database. There may be a variant in some tissue at E15 that is not present in differentiated ES cells. Also, you cannot say that E15 is the same stage as your differentiated ES cells. The embryo develops much faster in vivo, is likely to be much more complex, and have many more specific cell types than your in vitro differentiated ES cells. The second reason may be antibody cross reactivity to another related protein.
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