There may be a caveat to the above responses - if the person who produced KO mice was aiming for a functional disruption in the gene, it is possible to have a non-functional protein being synthesized and detected by Ab (if the antigenic epitope is different from the altered sequence in the gene). Such changes are possible by altering one or a few amino acids, for example in biding sites, modification sites or active sites of enzymes.
Without additional information about how the KO was produced, readers can only guess...
No. If you still detect protein expression (via Western blotting), you must have a cross-reactive epitope.
I agree on Mark Farman.
As an addition, it might also indicate that the gene silencing process is failed.
There may be a caveat to the above responses - if the person who produced KO mice was aiming for a functional disruption in the gene, it is possible to have a non-functional protein being synthesized and detected by Ab (if the antigenic epitope is different from the altered sequence in the gene). Such changes are possible by altering one or a few amino acids, for example in biding sites, modification sites or active sites of enzymes.
Without additional information about how the KO was produced, readers can only guess...
Good question and not so easy to answer. IN principle I agree with Tausif in that you may try to find wether there is just a fragment of the protein what is expressed; hopefully the not "functional". In any case you do not have a real KO as the truncated protein may do some "unnatural" effect.
http://www.biotechniques.com/BiotechniquesJournal/2013/March/Knockout-mice/biotechniques-341088.html
Same problem for me. I see a protein band in tissue from deletional mutants (2 different lines from two different labs targeting two different exons) using three different Abs from three different antigens, two species, and three suppliers. All Abs detect the base protein (~15 kDa) as well as reported multimers (~30 and 60 kDa). Seems a rather unlikely coincidence. Both labs report positive genotyping data, but no western data. PI from one lab says they're not convinced any available Abs are specific for the protein.
Unfortunately this is nothing new and may happen quite often or not, i.e. such "findings" may not be ever reported. In one case we were critized because a commercial antibody was not specific as it gave signal with a KO animal. Indeed the animal did not express the full protein but it did express part of the protein (no doubt about it). Novel strategies exist to be sure of complete lack of expression of a given protein but not all KOs are KO for the whole protein but express a shorter version. Are those real KOs (not in my opinion)
Pamela ,
I uderstand that you think that KO expressing a fragment of the protein is a functional KO.
If this interpretation is correct I would not agree because you would end up working with a new animal that does not express a full length protein but expresses something that is not expressed in the control (wild type animal). Perhaps we need a name for this "transgenic" animal that expresses proteins that do not exist in nature. In other words, should a scientist work with an animal deficient in a protein but expressing a "non-natural novel" one?
Same problem here. I got to work with previously made KOs. Three different Abs tested and none working properly. Finally, I found one that appears to work fine and voila! : all cells supposedly KO blatantly express the protein (full size). However, when I PCR'ed gDNA from those cells and cloned into a T vector and sequenced, all 10 clones showed a mutation. Could this go worse? Actually yes: ALL 10 sequenced clones showed exactly the SAME mutation!!! what are the chances of CRISPR-induced NHEJ repair making the same change in both alleles????? Nobody trusts my antibody because of 10 sequences. Is that enough?
It turns out that it is more common than expected that KO is expressing a fragment of the protein. If there is another explanation I would appreciate help.
Check this paper out. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178700.
It seems that protein can still be detected in KO cells then it is sure that RNA expression will also be there. But is it possible that your KO cells shows significantly higher expression (by qPCR) of KO gene than WT cells in virus infection ? While interestingly the KO cells do not show any change in the expression when treated with their specific ligand. Any help is appreciated. Thank you
Is it possible that only one allele is knocked out, while expression from the other wildtype allele compensates or is even boosted for some reason?
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