I'm doing a Western blot and haven't seen any protein for two months.
After transferring, the marker is visible, but when I detect the protein once, wash it, and try to detect another protein the next day, the marker is faint and the protein doesn't come out.
Some days, the protein isn't detected from the start.
I tried using Ponceau, but some days, a small amount of protein was produced, and some days, no protein was produced. So I checked the membrane of another lab member, and that friend was producing good results. I also did a mycoplasma test on the cells. Nothing was detected. I use the same lysis buffer.
I thought it was a problem with the washing buffer, so I made a new one, and I made all the solutions again, but this problem kept happening. Does anyone know what the problem is? I don't know what the problem is.
Dong Kwan Yoo Hi!, I have a few suggestions that might help.
1) The transfer conditions might be fluctuating. Please see if the power pack is maintaining a good current supply. Also at the same time, try looking into the transfer buffer.
2) Do you have any internal control like GAPDH or Beta Actin, which might help to know if the proteins are present in the lysates?
3) Does your SDS-PAGE still show proteins?
- Do you usually quantify your protein using the Bradford Assay before applying it to SDS-PAGE?
- Perhaps the protein extraction protocol doesn't suit your type of protein.
Chriswin Saimon Hi thank you for your kind reply.
yes, I already try run same time and same buffer for transfer but still happen.
and I also used actin and gapdh but also did not come.
Sâmera Breves Thank you for your kind reply. Yes! I always do Bradford Assay before applying it to SDS-PAGE.
Dong Kwan Yoo Even after using GAPDH and Beta-Actin, if you are not able to visualize proteins, I believe that your protein of interest might not be there in your lysates.
Also, I agree with Sâmera Breves saying that the protocol for lysate preparation might be different for your protein.
i've had some silly things mess up my western. check the pH of your wash buffer and Ab dilution solutions. my wash buffer was 4.2 for some reason and kept removing all my protein. also check that you are adding the right dilution of detergent to wash solution.
the best way to identify a protien molecule can be done with help of molecular diagnostic tools such as ELISA (sandwich, Direct and Indirect) or Influorescence or use of tools like molecular beacons or scorpions etc., The most efficient way to quantify protein, in my opinion is Lowry's Method to quantify protein when you are pursuing a research. Lowrys methodology is highly sensitive detecting even slightiest concentrations of protein which is not observed in Bradford. However, if you want simplicity (Lowry's procedure have steps) and if you think concentration of protien will be above 10 µg/mL, then bradford is the most suitable assay.
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