Hi,
I have problem detecting specific proteins using Western Blot. I am pretty sure that it is not the problem of my technique or antibody since I can still see loading proteins and ladders on the blot.
I am assuming that protein expression of interest is too low to be detected on the blot in the cell line I use. The protein is tumor suppressor and I was able to see the upregulation of this RNA after histone deacetylase inbhitor treatment.
When I run PCR these RNA start to show up around 27-28 amplification cycles. I tried to load lysates from 20-40 ug but I was still not able to detect the protein of interest using western blot.
Have you experienced this problem? What is the standard amplification cycle of RNA in qPCR to be detected on Western Blot?
If I cannot detect on Western Blot, is there any alternative to detect or support the low expression of these proteins?
Hi Bumjun,
it is very difficult to relate qPCR experiments to Western Blotting. They are two different approaches and depend on very different regulatory mechanisms. Expression level and stability of mRNA are one thing, translation efficiency and stability of a protein are a completely different thing. One could say that, in general, the higher the Cts, the less abundant will also be the protein, and this seems to be your case. A Ct close to 30 indicates a relatively low level of the mRNA, which makes sense for a tumor suppressor in a cancer cell line...
So, alternatives to the WB, if you are sure on that side you have done everything to optimize:
1) Immunoprecipitation, this will concentrate your protein, but you need to have an Ab that works... you have to look into this yourself
2) ELISA, this is used for very low abundance soluble peptides, such as blood cytokines, or Abeta-42...
Good luck
Thanks Dr. Hayakawa. I will look into articles.
Thanks Dr. Boyl. I will see if I can use those method to detect the proteins of interest.
I would advise first to validate your antibody with a positive control in your Western: a cell type with known high expression of the target or overexpressing the protein.
If you don't see anything, you can try to increase the amount of proteins (I have gone up to 100ug, depending on the background of the antibody) or test another antibody (the commercial ones are often not as good as they tell you, unless it is a very well studied protein).
If the antibody is poor, there is not much to do to be honest. You can test different conditions (increase antibody concentration, change blocking solution, etc.) but it is rarely a miracle solution. We had cases where lowering milk concentration to only 3%, or switching to serum or bsa instead of milk, helped a lot for instance, but it is not easy and it doesn't always help. Maybe you'll get lucky. Basically, we only play with the blocking if we get a perfectly white blot, then you can "decrease" the blocking a bit until you see some bands at least.
One thing that helps also is looking into specific cell compartments. Are you working with whole cell extract? Is your protein cytoplasmic, nuclear, mitochondrial or at the membrane? Or even secreted? You can use different protocols of extraction depending on the type of protein. That should help a lot if your protein is not simply cytoplasmic.
There are also different revelation systems with various sensitivity. If you have a white blot, try switching to a more powerful/sensitive system (for instance classical ECL -> high sensitivity ECL or Femto). Again, only if your blot is not messy to begin with.
If all of that still doesn't work, then your antibody is probably just not good enough for detection by Western. In that case, as mentioned by someone else, you can try Elisa or IP + WB, although be aware that both techniques also depend on good antibodies...
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