I have amplified my plasmids in DH5-alpha bacteria and extracted and purified the plasmids. Then I transfected SW1116 colorectal cell lines with the plasmids, although I can continue to see the GFP shining by fluorescent microscope (my protein product is GFP-tagged). Recently I have been having a problem in detecting my protein of interest by Western blot (previously I had no problem in this case). Ideas why?
Did you check for the transfer of the protein onto the membrane and further check for the optimum concentration of the primary antobody to be added.
There are plenty of reasons not to get a signal for your protein doing Western blot.
1. Bad extract
2. Bad electrophoresis
2. Bad transfer onto membrane
3. Bad primary Ab
4. Bad secondary Ab
5. Bad substrate for peroxidase (ECL)
Any of these steps can be a problem in detecting the GFP-protein. If all these steps are fine in your case, I would think that you have transfected your cell with just GFP (check the plasmide and use Ab against GFP for yor Western)
Regards
The yield of protein in the transfected cell lines could be very low to be detected, concentrate the sample and load on the SDS gel. Further proceed with the western blot and develop by ECL method. Using anti GFP antibody would be a good check for expression of the protein.
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