I am recently extracting murine islets. I see most researchers recommend pooling islets from several mice. I just want to check expression of my protein of interest in islets as an initial stage of work but i don't have enough mice.
If the initial purpose is to just confirm if a protein is expressed in islets, it is not necessary to pool islets from multiple animals, provided the protein is relatively abundant,
If on the other hand, the protein of your interest is not very abundant or your detection method is mediocre, you may miss the target and incorrectly assume the protein to be absent.
The above comments assume that either your protein is exclusively expressed in islets or you have a very reliable method of islet isolation which yields negligible contamination with exocrine cells. Otherwise you have an additional level of complexity.
If you can generate a decent cRNA probe that can be used for mRNA detection of protein of your interest, you could perform a few in situ hybridization experiments that could give you information you need and also confirm where this protein is expressed. Of course, this method is also dependent on the relative abundance of mRNA - higher abundance translates into better signal/noise ration and better quality results.
Hi Dina,
I agree with Dr. Alam.
You can get a decent yield of islets from one mouse (~150-200). But again, it would depend on your method of detection : protein or message. Handpick islets (transfer picked islets to fresh media and again handpick, repeat 2-3 times to minimize the acinar contamination) and purify RNA using on column DNAsing.
Best
agree with both- for Western or RNA you should hand pick Islets to minimize contamination of other cell types. If you have a good AB for staining a pancreas section will be good enough. You can id the islets by morpholgy or costain with eg. insulin.
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