Hello all,
What should I do to get rid of non specific band on western blot?
1. I used 5% BSA in TBST to block the membrane (1h, at RT)
2. Polyclonal primary antibody dilution: 1:1000 (overnight, 4C), 5x washing, each time 5 min
3. Secondary antibody dilution: 1:1000 (2H, RT), 5x washing, each time 5 min
4. Amount of protein load: 50ug
I got the band for loading control, but I get non specific band for my target protein. Here, I attached an image of the membrane.
Lane 1: Marker, Lane 2: Control (untreated), Lane 3-6: Experimental, Lane 7: Marker
p/s: the band inside of the red box is my target protein
Thank you in advance!
I think you should increase the dilution of secondary antibody to 1:5000 because too concentrate secondary antibody can increase background. We are using this concentration and getting consistent results even we are reusing our antibody 3 times. Our mice and rabbit secondary antibody are from Cell signalling technology company.
If your antiserum is recognizing non-specific bands, one way to clean it up is to adsorb it with cell extract from cells that do not produce the target antigen.
I think there is some band visible, but the background is too high. Indeed, as suggested above, you could dilute the secondary antibody, pre-adsorb primary antibody (here is another trick: collect antibody after using it once on membrane and re-use this fraction next time, it could give you cleaner signal) as well as reduce the exposure time (I'm not sure, but I think you use HRP-conjugated Ab'', hence you could easily play with the exposure time).
I think that you load too much protein per well (50 ug/well ). Normally the WB can detect about 10- ng protein. I recommend to load 1 ug/well, additionally the color developing time should be around 45 sec. To get pre-absorbed Ab is not that easy I would do it only as the last choice. Looking carefully on your WB one must consider that the primary Ab are not that mono-specific and were prepared against non purified Antigen.
Thank you all for your opinion. I already reduce the concentration of secondary antibody to 1:10,000 and change the primary antibody to monoclonal. But still, I got the non-specific band. What should I do right now?
One possibility is that some of the extra bands are due to proteolytic degradation of your antigen. If that is the case, you will have to prepare new samples that are more intact.
My first thought was to decrease your secondary antibody concentration, but after reading several posts, you apparently have and you still have the same problem based on the image you last uploaded. I think it could be one of several things: first that your primary antibody is not working well with your cell line. Antibodies bind differently in cell lines, and your antibody may bind to various splice variants in your specific cell line. Secondly, sample prep may be a player since it looks like you have a bit of drag in you lanes and marker. How long do you boil your sample for, at what temperature and what concentration of laemmli and beta-mercaptoethanol did you use. The post above mentioned proteolytic degradation; did you use protease inhibitors?
I hope that helps!
hello Nadhirah, do increase your blocking time and decrease your secondary antibody conc. its too high. For eg. if your primary antibody conc. is 1:1000 then your secondary should be either 1:5000 as said by shivani or 1:10000 also. In case if you are still getting the same then reduce your primary antibody conc. also
Dear all, thank you for all your opinion. I already get my protein band, as I prepare the new samples and changed the BSA (blocking buffer) into the one that has already commercialized. Thank you! :)
I would investigate what is this protein. By sending the protein band to sequencing you can learn a lot, and test if your first ab are responding with it.
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