I'm trying to carry out western blot to detect some proteins of varying sizes (between 70 and 37 kDa). I'm working on plasma samples along with cell lines as a positive control, but each time I get the exact 70 kDa band pattern.
I usually work with nitrocellulose membranes and antibodies purchased from Santa Cruz . I use a semi-dry transfer system and at first I was suspecting that I may have some issues with my stripping buffer so I have changed it. After several trials I also quit detecting different proteins on the same membrane by stripping method.
But I still get the same 70 kDa band which may probably represent Albumin protein !
How shall I get rid of such band or such protein that may be masking my protein of interest ?
Are there any technical tips that may help with this ? or any " Dos and Don'ts " hints ?!!
hi
1- You must use a negative control cell line to confirm the band .
2- check your membrane just by secondary antibody ( anti mouse or anti rabbit-HRP conjugated)
good luck
I am really not sure if I got your question right, but you said you are detecting several proteins of varying size. This mean you should use several antibodies in several blot,right?
Do you usually get 70kDa band size even if the predicted size is not 70? if that is the case then I agree with Reza, use one membrane in parallel and just ad the secondary antibody ( not the primary) if you still see this band then it is not specific band .
Do you do dilution for your plasma? if not you might want to try different dilutions( 1 in 2 to 1 in 10) your sample might be too concentrated.
I am agreed with Aziz Fahaad .
If you have different target proteins then you have to use different types of antibodies targeting your protein of interest.
I guess whatever primary antibody that you are using currently is only able to detect 70 kDa protein.
Reza Hadavi
Thank you for your answer
OK, I will try to check the membrane using the secondary antibody as you have suggested and I will let you know what I have got
Regarding, negative control cell line , I don't somehow get why shall this matter !
Aziz Fahaad
Yes sir, I use different antibodies but every time I get the same band even if I am expecting a band at 37 kDa not at 70 kDa. Actually this makes me feel uncomfortable with the antibody that shall give me a band at 70 kDa , I mean I become suspicious if this is my real band or its false positive one.
As I mentioned before, I used to try my first primary antibody then I strip the membrane and try the other primary, but finally I have quit this method. Now I use single membrane for each primary antibody
Regarding the dilution issue, I'm using Bradford assay to detect my protein concentration. The thing is that I get very high yields of proteins, so I tried several times to optimize the amount I will use for western to be 20 -40 ug and thereby I have made my dilutions , but I got nothing on the membrane. So now I perform western using equal volumes of my samples instead of equal amounts.
If you allowed me, this makes me ask, even if my samples are very concentrated why shall one band only appear while the other don't ?!
Md. Alamgir Kabir
Yes sir, I am using different antibodies that shall detect different sized proteins not only the 70 kDa protein.
Sarah Hamdy Hello Sarah,
To answer your last question , It might be because your secondary antibody is binding non specifically to any protein in your plasma sample . I think there is special preparation that is needed for your sample before you perform your western blot. please check the discussion in this link as it might hel https://www.researchgate.net/post/How_to_detect_protein_level_in_serum_by_western_blot
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