I have xenografted human tumor lysates that I am running western blots on. I need to determine the extent of mouse cell contamination in these lysates (I don't have slides for the tumors). I am looking for an antibody that exclusively detects a mouse housekeeping protein but not its human homologue on western blot. I have lots of antibodies that do the opposite: detect human, but not mouse version of the protein, but that doesn't help me much in this case.
I am thinking something along the lines of Tubbulin, GADPH, Enolase, Actin.
Hi Florian, not too sure that housekeeping genes will work. From my experience most companies try to generate for rodents only one AB that will detect as many species as possible (including humans). Maybe rather cell surface markers?
Do you still have some tissue left? You could extract genomic DNA and just run comparative PCR?
Antibodies with no crossreactivity with human species for:
tubulin - http://www.antibodies-online.com/antibody/93917/anti-Tubulin+beta+TUBB/
GAPDH - http://www.antibodies-online.com/antibody/728818/anti-Glyceraldehyde-3-Phosphate+Dehydrogenase+GAPDH+C-Term/
Enolase -
http://www.antibodies-online.com/antibody/1173051/anti-Enolase+2+Gamma+Neuronal+ENO2+Biotin/
http://www.antibodies-online.com/antibody/1174607/anti-Enolase+Muscle+Specific+MSE+Biotin/
http://www.antibodies-online.com/antibody/1174636/anti-Non-Neuronal+Enolase+NNE+Biotin/
http://www.antibodies-online.com/antibody/1077988/anti-Enolase+2+Gamma+Neuronal+ENO2/
http://www.antibodies-online.com/antibody/1077984/anti-Enolase+Muscle+Specific+MSE/
http://www.antibodies-online.com/antibody/1077991/anti-Non-Neuronal+Enolase+NNE/
Actin - http://www.antibodies-online.com/antibody/1113001/anti-Actin/
If you are looking for western blot, mouse monoclonal GAPDH is really a good one. Also the band is small in size so most probably it will not disturb you bands of interest.
Just had a quick look and e.g. human GAPDH isoform a is about 93% IDENTICAL to mouse GAPDH on amino acid level. The longest strech of non-identical amino acids is 2! I have no idea how the companies have created antibodies that only react with mouse and not human and indeed they don't say so, they only say it reacts with mouse and cross-reacts with rat.
So if you really want to go down this road, best control your ABs really well, for example on a human cell lysate.
protein Sca1 only in mouse.
http://tecnologia21.com/26134/inyecciones-contra-obesidad
Because you have already many human specific antibodies that recognize only human protein, you should be able to detect a house keeping gene from human. Purchase the protein from the house keeping gene from human and mouse. Run mouse protein as negative control to be sure it is not detecting mouse protein.
In the another blot, load a small standard curve of protein with different ratios of mouse and human, starting with only human, and doing 1:1, 1:2, 1:3, ... ratios with mouse house keeping gene protein.
In the same blot, load one of your samples alone, then load also your sample with different concentrations of the protein you bought 1:1, 1:2 and 1:3 ratio with (human:mouse) house keeping gene protein.
If you have the curves, you should be able to determine the degree of mouse contamination in your samples.
Good luck!
Hi! I'm facing a similar problem : I want to use an antibody that would detect a RAT protein but not its human counterpart. Any idea? Florian : any development since last July?
Hi, would you please to let me know the list of Ab that exclusively detects a human housekeeping protein but not its mice homologue on western blot? (except vimentin, HLA and mitocondria protein)
I am a kind of frustated in looking that.
Cheers
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