Hi,
I'm trying to examine the insulin and glucagon expression in immortalized pancreatic cells (MIN6, INS1, alpha-TC1-6). However, I am only able to get a very week signal - the samples look like the control. Any suggestions on what to do?
My protocol have worked on all cell types so far, and i testes with several different concentration of both secondary and primary antibodies. Could it be the cultivation of the cells that influence the intracellular protein concentrations of glucagon and insulin, the antibody binding or something else?
Any suggestions on what is wrong and how to fix it will be appreciated a lot!
Kind regards,
Christine Christiansen, Research Assistant, Aarhus University, Denmark
Thank you for your answers. Jeseph Papamatheakis, what would you recommend as positive control? RT-qPCR and radioimmonoassay already confirmed that the cells express both insulin and glucagon.
I suppose a validated cell line . Also you may run a Western blot if your antibodies are good for that. Where are they comming from? You may confirm your data with RTPCR although you cannot quantitatively correlate
I assume that the cell line is valid, as the expression have been confirmed by RTPCR, and that the problem may be the staining protocol. Is there any chance that the cultivation of the cells (for example the glucose concentration) could affect the protein expressions? My department unfortunately do not do WB, but I might be able to do it at another department, thanks for the suggestion.
Fothergill LJ, Ringuet MT, Sioras E, Hunne B, Fazio Coles TE, Martins PR, Furness JB. Cellular and sub-cellular localisation of oxyntomodulin-like immunoreactivity in enteroendocrine cells of human, mouse, pig and rat. Cell Tissue Res. 2019 Feb;375(2):359-369. doi: 10.1007/s00441-018-2921-z. Epub 2018 Sep 27. PubMed PMID: 30259122.
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