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CCRK (CDK20) protein expression and western blot


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7 replies to this topic

#1 kblee

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Posted 13 November 2012 - 06:32 PM

Hi guys,

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!

#2 bob1

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Posted 13 November 2012 - 11:58 PM

So what have conditions have you tried for the antibody detection?

Have you checked the sequence of the DNA in the plasmids?

#3 kblee

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Posted 15 November 2012 - 07:26 PM

Blocking condition: 5% BSA or 5% milk, room temp for 1 hour
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.

Thanks :D

#4 bob1

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Posted 16 November 2012 - 11:28 PM

Check that the antibodies you are using are suitable for western blot - antibodies against native proteins may not detect denatured forms.

#5 kblee

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Posted 18 November 2012 - 11:42 PM

Thanks! Those three antibodies are all suitable for western blot.
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#6 bob1

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Posted 18 November 2012 - 11:59 PM

Can you try detecting it in another cell type, such as HeLa or 293 to see if this is a problem with protein expression levels? Myc antibodies are pretty specific and will detect at quite low levels, so it is possible that there just isn't enough protein there to see with your antibodies.

#7 kblee

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Posted 19 November 2012 - 12:04 AM

Can I know which antibody you are using to detect the CCRK? Since CCRK has 4 isoforms, do you know which antibody can detect the longest isoform? And which cells you use to overexpress CCRK? Thanks so much!

#8 bob1

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Posted 19 November 2012 - 12:06 AM

Sorry, I don't work with CCRK, I'm just offering general advice for any transfection/western detection problem.




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