I want to detect my target protein through WB and it worked well before. But now I could not get any signal for both my target and the GAPDH, so I ran SDS-PAGE. I expected many bands on the gel of my cell lysate but in fact there were not. I was wondering that if it is normal to get the result like this (see attached). Is it because even though there are many proteins in the cells but the amount of them will not be big enough to give obvious bands on SDS-PAGE?
I do not know what exactly is loaded here, but if you say that there is no detection in the WB of either your protein or control protein (GAPDH) then I would simply assume that there is no cell lysate in your sample since WB shout be a very sensitive method for detection of protein.
Or, there is cell lysate but the sample underwent some treatment that impaired any downstream analysis. Do you have a comparison of lysate tested on SDS-PAGE from before? Because I agree, these samples on SDS-PAGE do not look like lysate samples. Or your load is simply very very little since there is some kind of faint smear.
My best guess is that the samples were mixed up, the harvest not performed correctly or the sample was treated in a way that makes it not compatible for downstream analysis.
Hi,
I think there are some potential reasons why you might not see bands on your gel:
I hope the information is useful for you.
Good luck!
Thank u for the advice. Actually, I have not done SDS-PAGE of the cell lysate before because I did WB directly and got positive results, which means all parameters were optimized properly. Since last time I could not find my target bands, I firstly performed SDS-PAGE to test the overall proteins in cell lysate (loading 13uL lysate sample/well). PMSF was added when I lyzed the cells and kept it on ice. So I want to know that if anyone have done SDS-PAGE of cell lysate and visualize it. What kind of bands were obtained? What does the result look like? For example obvious bands or smear? Phan Thi Lam Hong Philipp Czermak
Hi, could you please specify the amount (in micrograms) of lysate loaded per well?
Hi,
To accurately determine the loading amount, it's essential to measure the protein concentration of the lysate. The amount of lysate loaded can vary depending on the specific targets you're detecting. Additionally, the quantity of lysis buffer added depends on the number of cell pellets. If you aim for a higher lysate concentration, reducing the amount of lysis buffer is advisable, and vice versa.
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