Hello, experts.
I have been struggling during my Western blot.
Briefly, I transfected my gene into two different breast cancer cell lines. The introduction of my gene triggered cell death in one cell line but not in the other cell line. So, I tried to check my gene expression at the protein level. In my western blot, I successfully detected my protein (flag tagged) from the living cell line but no signal was detected from the dying cell line. I have tested different time periods after transfection and protein isolation methods, for instance including or excluding dying cells. It is not the problem in the vector construct because I can detect the protein from the other cell line. It is also not the problem of the cell line because the positive control vector which shares backbone with different insert was translated successfully. is there someone who knew what happened?
Hi,
My suggestion to you to transfect your gene into another two different breast cancer cell lines but this time you need to use a housekeeping gene that has a stable and similar expression in both cell lines to measure the expression of a gene properly. After that, you can use you can proceed with qPCR.
Hope this may help you.
One can detect protein expression in cells by immunofluorescence. If not possible, consider the possibility of recovering protein of interest from the culture medium.
Hi Kang-Hoon Lee,
Please check the protein isolation and quantification method that you have used. You may note that if your protein extraction method contains detergent such as SDS, you are not advised to use Bradford method as it may affect your result (total yield). Therefore, please use an alternative method such as BCA or Lowry method. As concentration of your total protein that you have isolated from the source (necrotic/apoptotic cells) seems to be very low, and moreover, if you are using a protein estimation method that is incompatible, you might apparently detect no proteins at all. For assessment of different mitochondrial complex enzymes in cells exposed to various stress stimuli, we adopted the above described methodology and obtained satisfactory results in both WB/ELISA experiments.
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