From the primary hepatocyte culture of C. batrachus, I am getting protein concentration in the range of 7-10 µg/µl in Bradford assay. However, in western blotting, I use to load 50-75 µg total protein in each lane. I have used a range of primary antibody concentration from 1:2000 to 1:10000 but, still was not been able to get any band for mostly abundant proteins. In case of endogenous control also (GAPDH), a very faint band is coming. So, what might be the reason behind this or what should I follow to get result in WB. Moreover, what is the usual concentration of protein one gets from a primary culture?
Hi, I recommend you to check these points:
First, the composition of your Transfer Buffer and the percentage of Methanol. Then to ensure whether the proteins have been transferred or not, stain your gel with Panceau. If you could detect sharp bands there might be something wrong with your transfer buffer. Also, do not forget to activate your membrane if you use PVDF. Moreover, the percentage of skimmed milk is very important. And finally it is better to incubate 24 h in primary antibody.
Wish it helps you...
Hi,
I recommend you to check these out:
1. Composition of your Transferring Buffer
2. Percentage of Methanol in Transferring Buffer
3. Stain your gel with Panceau to ensure that the proteins have been transferred
4. Ensure that you have activated the membrane if you are using PVDF
5. Check the percentage of skimmed milk for blocking buffer
6. Incubate primary antibody for 24 h
Wish it helps you
Western blotting is a relatively complex procedure, and possible causes for failure may occur at different steps. (i) Are you sure that you got a good protein transfer to the membrane? (ii) Have you titrated your primary antibody? (iii) What about the secondary antibody (or whatever you use for detection of the bound primary antibody, e.g. protein A or G)? - The 1° antibody concentrations you indicate seem pretty low to me. For comparison, in order to detect HIV antigens in lysates of virus-producing cells three decades ago, I used serum of AIDS patients at a dilution of 1:25 to 1:100 and secondary goat anti-human IgG at 1:1000. In order to use such low 1° antibody concentrations as you describe you need pretty powerful monospecific or monoclonal antibodies.
I have used a stain free gel and thus have taken picture of the gel after running. There also I have found very faint bands. After that I have transferred and stained the membrane (Nitrocellulose membrane) with Ponceau. But there also I got few very faint band. I have made loading sample for WB with concentration 5 µg/µl and loaded 15µl so that total protein in each lane becomes 75 µg. Why no band is coming instead of loading 75µg of total protein? Moreover, in case of tissue, I use to get very convincing band with the same amount of total protein.
Dear Debaprasad, you write "Moreover, in case of tissue, I use to get very convincing band with the same amount of total protein." Does that mean that your problem arises when using something else? Please identify the kind of sample you use, otherwise you cannot expect to get a useful answer.
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