Would anyone recommend a specific antibody and/or target to co-localise a protein in the secretory pathway? Would you recommend targeting the rough endoplasmic reticulum, if so which antibody (as I had problems finding any such antibody)?
Would it be possible to perhaps detect endoplasmic reticulum using e.g. ERp72 or Calnexin and at the same time stain for ribosomal markers to get the rough ER?
Any other suggestions to show that a protein is being secreted by looking _inside_ the cell is warmly welcome. :)
Thank you,
Josephine
Depends on your protein of interest, but I would suggest the specific protein of interest rather than the organelles involved. If you know the amino acid sequence there are numerous companies available that can generate specific antibodies to the protein. I would suggest different length moieties of the same protein. In that respect you could track your protein as it is being generated. One of the keys to using antibodies is to understand how to get the antibody into the cell, if the epitope is intracellular. We have used tween-20 as a method to get the antibodies into cells. Another detergent that could be used is CHAPS, although tween-20 is less harsh on the cells.
Hi Josephine,
I second Henry's points above regarding using targeting the protein of interest rather than the organelles involved. I couldn't get from your question whether you do have an antibody for detecting your protein of interest? From its subcellular localization you might get some hints about the organelles involved.
Cheers!
Hi Josephine,
I'm not aware of a specific antibody for the rough E.R. But common markers to co-localize by immunofluorescence microscopy proteins with the E.R are calnexin, PDI and calreticulin. Although if it localizes with E.R or not it does not mean it is or not a protein that follows the secretory pathway. It can associate with the E.R (but be cytoplasmic) or it can be made and quickly traffic to its final destination and you will not be able to see its steady state in the E.R but maybe in some other compartment of the secretory pathway. So if you are not at all aware of its location in the secretory pathway you should consider markers for other organelles.
You should also look in the sequence of the protein to check if there is a signal sequence to target translation to the ER or if the protein as any transmembrane domain. You can also try cellular fractionation.
Good luck
Hi all,
Thank you for your responses. I should probably clarify a few things:
We are going to secrete a protein in human cells that is not normally secreted, by fusing it with a secretory signal peptide in its N-terminal. We are looking at some different options and would like to compare the efficiency of the signal peptides to actually lead to secretion.
As we are in the beginning of our studies, we want to ensure that the protein is not targeted for degradation, and of course another natural way is to look in the supernatant of the tissue culture, and this will be done at a later stage. My question was only aiming to find out if I can colocalise the protein with the secretory pathway. I had found out about Calnexin and PDI, and Sonia has also added calreticulin to the mix, thanks!
If anyone has a new revelation of what to do in our case, I would kindly recieve your answer. :)
Ta!
Hi Josephine,
The 1st thing I will do is to look at the supernatant of tissue culture to check if your protein is secreted as it is the most reliable test. I'm checking for secreted proteins upon transcription activation by a transcription factor involved in secretion in Liver and have identified proteins in media upon precipitation. It is very easy to get.
If your protein it is not usually secreted the most likely is that it will be recognized in the ER as misfolded. But maybe a lot of it will be able to do its way to the exterior. If you are just interested in getting the protein outside I'll not care about its degradation. If somehow you still want to get an idea of how much it is degraded you can do a cyclohexamide chase and if you are not able to detect your protein in cells you can add an inhibitor of the proteasome to block its degradation (this will also give you an answer of whether or not the protein is degraded).
Good luck
Sonia
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