I’m new to the field of immunology and don’t have any prior experience working with neutrophils.
I’ve been isolating neutrophils from whole blood, lysing the cells directly after isolation with RIPA buffer (including protease and phosphatase inhibitors), and running the samples on a 10% SDS-PAGE gel. However, I’ve encountered some confusing results. Specifically, I’m not getting any signal for actin as a loading marker, which is even more puzzling than the inconsistent results I’m seeing with my target protein band.
I’ve tried using different actin antibodies as well as samples from different donors, but both times I didn’t observe a band for actin. Interestingly, other bands appear when probing with the target protein antibody, so I know there is lysate in the well.
Do you have any idea why this might be happening? I’d greatly appreciate your help.
Are you using the antibody for the first time or someone has used it before? if it is the first time, make sure it targets a correct antigen i.e if neutrophils are from human then anti-human the same to your secondary antibody if the primary antibody was raised in mice then your secondary antibody should be anti-mouse.
Hi Thomas, been using this antibody for many years works everytime with human cells and samples. Also, in the same blot along with neutrophil lysates I have also other cell lysates and it detects actin fine. Its only in the lane which has neutrophils that it shows empty.
What is the cell count and protein loaded per well? Try to have good number of cells and optimum protein amount to be loaded per well. You will surely get actin bands.
I had 52 million cells and BCA showed that I had around 2 ug/uL protein and I loaded 60ug per well. I think there was protein degradation as neutrophils expel a lot of proteases when activated. I have to find a way to avoid excess protein degradation.