Hi, it looks like high background. I guess the 1st well is having positive control but band is not as dark as background. There is hardly any difference between the transfected and untransfected cell lysate. First try to get rid of background. Did you use any detergent (tween20) and check the concentration. washing optimizations may help (rocking rpm, incubation time),

If you used to get single band and now this then what did you changed try to find out. May be your antibody (primary or secondary) gone bad, check with different lot or brand to figure out. you can do it with just positive/negative control and single immunoblot. Cut the blot in multiple small section and try with different antibody combinations without changing your current protocol. Santa Cruz antibodies work well but comes with expiry date sometimes as close as 6 monthes, try to use new lot.

Hi Suchi Chaturvedi . I would suggest getting your antibody from another company. SC is not known for making good antibodies.

It seems you also have an issue of insufficient transfer efficiency as even your standard seems fairly faint. Your multiple band issues appear to be from insufficient blocking or indeed low specificity of the antibody. Block with 5% Skim milk for 1 hour at room temperature. Dilute milk in TBS-T or Tween 20 washing buffer and yes as suggested before try a different antibody.

I'm thinking it's due to a change in the antibody batch as well. There can be huge quality differences lot to lot (my PhD lab always made note of which batch was used in case there were poor quality ones). What happens if you run a blot with un-transfected cells?

I assume you use secondary antibodies, but not conjugated version of primary antibodies. Have you tried using second antibodies raised in different species, e.g. anti-mouse raised in goat? Maybe your secondary is nonspecifically binding, if so, optimizing primary antibody conditions will likely not work. You can also consider shortening the time of second antibody treatment.

Hi, Suchi. in my view, this WB you showed is a successful experiment, so the problem may not be your skill of WB. first, I don't know the position of 34kDa, it will be helpful if you mark it. Second, if other bands are all lower the expected band, I will say your target protein may be degraded. To this problem, We often digest cells with trypsin and neutralize with complete media, spin for 300g, 2min , and wash one time with PBS. at last, the pellet of cells will be lysis with RIPA for 30min in 4℃ , usually add DTT & PMSF in RIPA freshly(both 1mM), it is the key to keep your protein from degradation. Third, if other bands are lower and higher than target band same time, I think your first antibody lost its specification to your portein. This may be caused by recycling of first antibody. Many labs will recycle first antibody, but that's under the premise that the antibody is used in 4℃, and do not freeze thawing more than 3 times. It will be helpful for preservation of antibody in 5% BSA, and storage at -20℃.

Hi Yunpeng, Thank you for the suggestion. Here I am attaching a picture of the blot marked with protein size. I absolutely agree with your second suggestion and implicate it in the next round of WB. But Here in this blot, I used fresh antibody and the blocking was done with 10% BSA for 3 hr at RT and antibody incubation at 4 C in 3% BSA O/N. Still, I am not getting signal band (previously I used to get signal band but from past one month I am getting multiple bands including in control (1st and 5th lane only cells)