Hi,
I am doing western blot on the murine Gfi-1 protein and have a question regarding to that antibodies I use. I have used two antibodies, one from Abcam regognizing the C-term part of the protein and one from SantaCruz, regognizing the N-term part. Both Abs are working fine, but the bands I get with the Abcam and SC antibodies are different sizes - 49 kDa and 55 kDA, respectively - How can this be? Both companies state that these are the correct sizes.

1. what is the size of you target?
2. did you use the same PAGE system for both WB? sometimes the size of proteins will be altered by different gel systems?
3. you may have unspecific binding

My protein is 46kDa but Abcam says that it migrates as a 49kDA protein (http://www.abcam.com/index.html?datasheet=21061) and SantaCruz says that it migrates as a 55 kDA protein (http://datasheets.scbt.com/sc-8558.pdf). I have used both antibodies on the same WB membrane so the conditions for the gel are the same. The bands dissapear in both cases in competition with a peptide so they are both specific.

although I personally trust better SantaCruz AB, and you may have already noticed that the SantaCruz AB have some really good citations.

to make sure, in this case, strongly recommend
1. run a positive control if have one. if not,
2. get both bands from your protein gel and send for sequencing


hope it helps

I don't have a positive control...It is not available unfortunately (or at least I haven't benn able to find it). I will look into the sequencing...
But if both the 49kDa band and the 55kDA band are the wt form of my protein....is there a logical explenation for this? I don't understand how the same form of a protein can give rise to two different bandsizes.

Thanks for your help :)

it could be that one protein appeared two bands on a gel, they could be differently charged, phosphorylated, glycosylated etc.

but if they are the same, both of your ABs should detect 2 bands