I am currently working with a peptide which is an analogue for glp-1, but during invitro studies am not able to detect for the presence of GLP1- receptors. The cell line used is Min-6. How do I detect it?
3
-
$\begingroup$ If you have an antibody against the GLP-1 receptor, you could make a whole cell lysate and do a western. How old are your cells? According to this they alter their behaviour over time and alter the expression of genes. $\endgroup$– Chris ♦Feb 13, 2014 at 14:30
-
1$\begingroup$ Try polyclonal antibodies. It might just be that your antibody isn't picking up the epitope. $\endgroup$– dayuloliFeb 14, 2014 at 5:43
-
$\begingroup$ does the cell line express GLP-1 mRNA? You could easily and cheaply use PCR to amplify the GLP-1 mRNA. That technique would be exquisitely sensitive and specific for what you want - and you don't need to worry about expensive antibodies. $\endgroup$– Vance L AlbaughNov 24, 2015 at 15:31
Add a comment
|
1 Answer
$\begingroup$
$\endgroup$
The question is slightly unclear since it fails to clarify whether the GPL-1 receptor is endogenous or is over-expressed. Also does the GLP-1 belong the same species that Min-6 originates from or it belongs to a different species? Those are important points since if the receptor is endogenous then the main problem is sensitivity of the detection method, which can be difficult sometimes to detect an endogenous protein relative to an over-expressed one. Also if the GPL-1 belongs to a different species, which you are trying to over express in Min-6 it might not work since the cellular machinery might not support its production (tho unlikely) or its transport to the cell surface, which I have had this problem before. It could be that for the particular expression levels you have in those cells, if the GLP-1 is over expressed or if its non-endogenous, it causes toxicity and kills the cells that are expressing it hence why you do not see any GLP-1 expression (e.g due to excessive receptor activation). Also could you clarify what you mean by "analogue for glp-1"? does this mean it is not exactly GPL-1 or its a homologue or orthologue, which you are trying to detect using a GLP-1 specific antibody? that might again be a problem if the GLP-1 Ab you are using detects a region of the receptor, which is not conserved in its homologues.
In terms of solutions as pointed in the comments, the best way to go about this is to use a polyclonal Ab, which is raised against a large section of the peptide in question, allowing you a better chance of its detection. If that did not work, you might have to resort to GLP-1 mRNA detection using RT-PCR or qPCR, which you would need to design primers for based on you GPL-1 sequence which you can get help with here.
answered Aug 14, 2014 at 8:10