Hi all.
I have western blotting twice to see the expression of GFP in HeLa cells, but unfortunately both times I saw band for GFP at ~ 44kDa ( we expect at ~25kDa) and a band for TDO2 protein with HA tag at ~ 44kDa (here we are not expecting any band) because the primary antibody we added here is Mouse GFP and the secondary antibody is Goat anti-mouse IgG HRP. Can someone explain the possible reason for these weird bands? I have attached the image of WB.
Thanks in advance,
Keerti.
Probe your blot either with primary against HA or TDO2 that solves the problem.
All the best
Hi,
Firstly, If you wish to see the expression of GFP the ideal way is to see fluorescence by microscopy. Are you sure the band you see under GFP is genuine. You have a huge background and it doesnt seem like a genuine band.
Also from your question it is hard to understand if the signal for TDO2 was obtained using antibody against which target protein.
Hi Keerti
Check if you are using GFP (~25KDa) or EGFP (~40KDa) and if you have any tag in the protein sequence.
If you blot only for GFP then the lower band is a GFP degradation product or just a non-specific band.
Keep in mind that antibody might cross-react with other proteins if the blocking is insufficient or the concentration too high. Also at higher exposure, like it seems to be the case, you will see non-specific bands.
As already suggested you should check the expression of GFP by fluorescence microscopy and you should blot two separate membrane with anti-ms-GFP or anti-ms-HA only.
Best,
Edoardo
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