I recently carried out ELISA on L6 skeletal muscle cell lysates using Elabscience GLUT 4 Elisa kit in order to determine the GLUT 4 expression in the cells when treated with different treatments. However, I have been getting no expression in all my treatments including the positive controls although, my standards for the Elisa produced a good curve. I tried to use different methods to extract the cell lysates and i confirmed that the treatments were done properly but there isn't any difference when I repeated the ELISA. All the results from the ELISA appear negative. May I know if anyone has used ELISA before to determine GLUT 4 expression in these cells? Which extraction method was used? or perhaps is there another better method to determine the expression?
Jacinta:
Regarding your question, I think the experimental approach you choose will depend on your research question.
If you want to evaluate gene expression, I would recommend using RT-qPCR. On the other hand, if you are interested in looking at the protein abundance of GLUT4 in lysed cells, I would recommend using Western blotting with GLUT4-specific antibodies.
On the other hand, if your experimental conditions or controls include insulin stimuli and you want to look at GLUT4 translocation, you should use a cell line that expresses GLUT4 with some epitope. This is because commercially available antibodies are not very sensitive in distinguishing between GLUT4 and other GLUT isoforms. For example, Dr. Amira Klip has developed a technique using a type of L6 myoblasts, which express a "myc" epitope in the first exofacial loop of the protein. With this technique and using a myc antibody, you could detect surface GLUT4 levels in fixed and unpermeabilized cells by immunofluorescence.
Here you will find more information about GLUT4myc cells:
https://www.kerafast.com/item/840/l6-glut4myc-rat-myoblast-cell-line
Regards!
Javier Russell-Guzmán Thank you for your response. Yes I am also doing western blotting for the GLUT 4 as I am mainly looking to determine its quantity in my samples after treatment. The part about the GLUT 4 translocation is also really insightful and helpful, I will read more about it. How about the ELISA though? Is it not recommended to use for determining the GLUT 4 quantity in skeletal cells?
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