My protein of interest has a disulfide bonds, and it's a transmembrane protein. However, studies showed that this protein localise not only in the plasma membrane, but also found intracellular associated with other organelles. We are aiming to recruit the PDI antibody for the above purpose, but I'm not sure to what extent that this approach is feasible? and whether using this antibody by following a previously successful methods might be the best?
NB: there is no commercial antibody against this protein, so that's why we are looking for an alternative ways.
Any further contributions is much appreciated.
Ibrahim
PDI is protein disulfide isomerase? That's an ER resident, if your protein is in the ER you should be able to see that by morphology alone. If you can't find antibodies to PDI, why not use BiP antibodies (an ER chaperone)?
But you should use other markers as well besides the ER, one for the Golgi, one for early endosomes, one for late endosomes, one for recycling endosomes, one for lysosomes.
Thank you Jürgen!
Yes, PDI is stands for protein disulfide isomerase.
I do have antibody for the PDI, but how I can see that by morphology as you mentioned? Is there any difference between the BiP as marker and the PDI?
It's very interesting and essential to have the other markers. Please, could you suggest which marker could work the best for Golgi, early endosomes, late endosomes..etc?
ER staining means you see the nuclear envelope and a sharp reticulate network emanating from there to the edges of the cell. It is easy to find images in standard textbooks (Molecular Biology of the Cell). Post-ER compartments are usually punctate and harder to distinguish, although in mammalian cells the Golgi stacks are all clustered together quite close to the nucleus and for a very bright fluorescent blob. ERD2 (ER retention defective 2) is a Golgi marker, EEA1 (early endosome 1) is an early endosome marker, Rab11 is at the recycling endosome, I would start with those three if your staining additional to PM is punctate.
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