CCRK (CDK20) protein expression and western blot
Posted 13 November 2012 - 06:32 PM
I am facing a problem with overexpress CCRK in HCC cell lines for long enough. The plasmid cannot overexpress this protein in HCC or Liver Normal cells.
To solve the problem, first I made a new plasmid with pcDNA3.1+ backbone, still see no difference. Then I clone the gene into a myc-tag plasmid. Although can see fusion protein when compare with control plasmid, but however no difference when probe with CCRK antibodies (Yes, it's antibodies because I bought 3 antibodies from abcam trying hard to probe them).
So anyone is working on the same protein or give any suggestions? Why the plasmids can make fusion protein with myc-tag but cannot detect by the CCRK antibodies? What can I do?
Thanks all of you in adcance!
Posted 13 November 2012 - 11:58 PM
Have you checked the sequence of the DNA in the plasmids?
Posted 15 November 2012 - 07:26 PM
Primary antibody: using TBST or in blocking solution for antibody, dilution from 1:100 to 1:1000 4C O/N
Secondary antibody: using TBST or in blocking solution, dilution from 1:2000 to 1:5000 RT 2 hours
Protein amount: 30ug to 100ug
Yes, the sequence of my insert gene perfectly fine. In frame and without any mutation.
Posted 16 November 2012 - 11:28 PM
Posted 18 November 2012 - 11:42 PM
Posted 18 November 2012 - 11:59 PM
Posted 19 November 2012 - 12:04 AM
Posted 19 November 2012 - 12:06 AM