Need Help with Western Blot Experiments

  • Thread starter Selvar123
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In summary: Selvar123I am using antibodies from three different suppliers: dCK, TK1 and PCNA. I tried them all and I could not get any band at all. My colleague could not get any band with PCNA antibody. I changed the reagent and still could not get results.
  • #1
Selvar123
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Hi Forum,

I have been working with Western blot for a while now. I got beautiful results in the beginning when I started the experiment. Now I am stuck with three more antibodies, which I have been trying for the last 5 months, and not getting any result. I could not get any band at all. I do not know what is going wrong. I took help from people in my lab who at least get bands from my own sample. I felt that there was something wrong with my reagents, but when I changed the reagent, even then I could not get results. Please, is there anybody to help me with my situation.

Thanks
Selvar123
 
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  • #2
Hi there Selvar, perhaps if you could outline exactly what reagents, antibodies, protocol etc you are using so that members could get an idea of what is possibly going wrong.
 
  • #3
Selvar123 said:
I could not get any band at all. I do not know what is going wrong. I took help from people in my lab who at least get bands from my own sample.
What are you saying here? That other people do get bands with your sample and antibody, but you don't?
 
  • #4
Hi Forum,

Thanks for the reply. I am trying to work with dCK, TK1 and PCNA antibodies. I could not get bands for dcK or TK and the band that I got for PCNA was not satisfactory, the PCNA is supposed to be a strong antibody, but it did not show sharp bands. This was the case with me as well as my colleague.

I use reagents prepared in the laboratory, consisting of tris-HCL buffer (running and transfer buffers), reagents for preparing gels such as 1.5M and 1M tris, 30% acrylamide, sds, 10% fresh APS, TEMED. I prepare 12.5% gel everytime. I load protein according to Bradford assay by plotting the standard curve, so that I have equal concentration of protein in each well.

Once I load the gel, I run it for 45 minutes and put it for transfer in cold room for 2 hours. I block the membranes with 5% non fat milk for an hour and incubate it overnight with primary antibody. the next morning, I wash the membrane 3 times and incubate with secondary antibody for 1 hour. I would wash the membrane several times and place it in ECL 1:1 diluted. Then, I would take the membrane for filming.

I DO NOT do the coomassie stain.

I hope this would help people to help me to troubleshoot. Please suggest if any other information is needed.

Thanks
 
  • #5
Selvar123 said:
the PCNA is supposed to be a strong antibody

How do you know this? Sometimes antibodies that work for one purpose (e.g. immunofluorescence) do not work well for other purposes (like western blotting). Are there antibodies from other suppliers that you can try?
 

1. What is a Western blot experiment?

A Western blot experiment is a laboratory technique used to detect and analyze specific proteins within a sample. It involves separating proteins by size through gel electrophoresis, transferring them onto a membrane, and then using antibodies to detect the desired protein.

2. What are some common troubleshooting tips for Western blot experiments?

Some common troubleshooting tips for Western blot experiments include ensuring proper protein transfer onto the membrane, using fresh and properly stored reagents, and optimizing the antibody concentration and incubation time.

3. How do I choose the appropriate molecular weight marker for my Western blot experiment?

The appropriate molecular weight marker for a Western blot experiment should be chosen based on the size range of the proteins being analyzed. It is recommended to use a marker with a wide range of sizes, including a band near the expected size of the target protein.

4. Can I use a Western blot to quantify protein levels?

Yes, a Western blot can be used to quantify protein levels by comparing the intensity of the band for the target protein to a standard curve generated from known protein concentrations. However, this method is not as accurate as other techniques such as ELISA or mass spectrometry.

5. How do I store and handle my Western blot samples?

Western blot samples should be stored at -20°C or lower to prevent protein degradation. It is important to handle the samples gently to avoid protein denaturation. Additionally, it is recommended to use fresh samples whenever possible to ensure accurate results.

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