Hello, I am trying to see and quantify by western blot the ZIP9 or SLC39A9 protein in Jurkat and Raji cells. I am using the parental lines and ZIP9-knockdown lines (by qRT-PCR I have observed an 80% decrease of ZIP9 mRNA). However, when developing the western blot, it is difficult to see the bands, and when I manage to see them, I observe no differences between the parental cells and the control cells. Cell lysis is done with RIPA Buffer + phosphatase and protease inhibitors and I use Abcam #137205 and Thermo #PA5-21074 antibodies. Can anyone give me an advice? maybe ZIP9 being a membrane protein, should I use another cell lysis technique?
thanks in advance!
Hello Miguel,
Since you provided the catalog number for the antibodies you used, I am wondering about their specificity. The Abcam antibody detects a protein at about 30 kD (image on webpage) while the Thermofisher antibody detects a protein at about 51 kD. This raises to me the question, whether both antibodies detect the same protein. Do both antibodies work comparable good/bad? The Thermofisher antibody can be blocked with a blocking peptide ( PA5-21074 can be used with blocking peptide PEP-1188), which could help you to identify the signal you are looking for.
Good luck!
Hi Gisela Beutner,
Thank you very much for your advice! With the Abcam antibody I have been able to detect 50 kDa bands, but I have seen no difference between wildtype cells and knock-down cells for ZIP9 (which I have confirmed by RT-qPCR). However, with the Thermofisher antibody I have not been able to see anything, I always get a blank membrane. I don't know what the problem is, maybe there are few levels of this protein, I am thinking of obtaining the protein by subcellular fractionation to see if I can see it better.
Hi Miguel,
I think you are in the right direction. Either your antibodies are not specific (considering Gisela's comment above and also that it gives signal even with the KO cells), or your samples do not contain your favourite protein. Validating the antibodies AND purifying the membrane proteins specifically should be your next steps. As most membrane protein are not easily soluble, you may need to do subcellular fractionation.
[Article Subcellular Fractionation
]To remain sane, first use another well-validated antibody for a membrane protein, to establish your lysis/purification protocol, and then try to detect your favourite protein.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes