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?
Greetings. I need to do lots of Native Chromatin Immunoprecipitation using both fresh or snap frozen liver tissue. Since the clinical samples are precious, we will use Medimachine to homogenize the samples to achieve the nearly single cell suspension status.
1. When using frozen tissue, suppose the cells dead. Can I merge the data using fresh tissue and frozen tissue?
2. Someone told me that there're tons of enzyme in the liver cells, when go through homogenizing procedure, the protein degrade extremely fast. So should I let the tissue go through the Medimachine procedure in frozen or let it thaw a little bit?
3. I know using tissue to perform ChIP is very difficult, not too much journal can be found in pubmed, why so difficult? Anyone has experience on N-ChIP using tissue can share the pain and joy?
I am working on a kinase that very difficult to detect by antibodies (try three abcam in N-terminal, monoclonal, or the most expensive one)
Since I have to over-express it in some cell lines, I just made a plasmid with myc-tag follow the kinase,so it became a fusion protein.
Yep, the band came out, but there is a very strong band in the all samples, e.g mock (transfection reagent only), parental cells, over-express and control vector one.
Another wired, there is a lower molecular weight band (like lower 10kda) appeared. I think the myc-tag should be specific to the fusion protein...only.
1: Anyone can tell me why a strong non-specific band in all cells?
2: Why there 's a smaller band appeared in western blot?
3: Anything I can improve in the western?