I am using a primary antibody (1:500; from cell Signaling Tech) which has been made in BSA. I am using 5% NFDM as blocking solution and then washing the membrane in 1X PBS for 10 minutes and then one wash of 1X PBST for 5 minutes. The bands of proteins like GAPDH and other 2 proteins are really good but the antibody made in BSA always has a lot of background and uneven dumbell like appearance. I tried to inc./dec. the secondary antibody dilution but nothing seems to be working. I have attached the image of the antibody i'm facing problem with. Please suggest some alternatives so that i can record some good pictures of this protein with less or no background?
After washing with IX PBS and 1X PBST i'm incubating the membrane in antibody made in BSA overnight. Then next day 3 washes with 1X PBST and then 1.5 hrs of sec. antibody followed by three washes again and then image development. To eradicate dumbell shaped appearance of the bands i diluted sec. antibody until 1:50000. This dilution made the dumbell appearance went away for other protein of interst but not this one.
Try primary 1:1000 and try to do blocking overnight at 4c with BSA (or blocking buffer from thermo) instead of milk.
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