I have a FLAG-tagged protein (no endogenous antibody so for now everything has to be done via over-expression)
I over-express in HEK cells, make whole cell lysate and run on a western
Probe with FLAG antibody and see two bands - one at the correct size and one slightly higher (~5 kDa)
Protein is predicted to be phosphorylated and/or glycoslyated
So I treated with de-phos and de-gly enzymes
Sure enough the upper band disappears after treatment
Except it also disappears in my controls!
The controls are treated the exact same ways but no enzyme added
Wondering how incubation at 37 degrees could remove the upper band regardless of whether any enzyme is added - what could this mean it is?
Am I getting too hung up on this? Is it just non-specific?
I just know that when I show this in lab meeting then everyone will want to know what the upper band is......
Re: Disappearing band
Hola, Check the reducer power of your loading solution. Moreover, prepare a non reducer with SDS loading solution, and apply the sample without boiling and compare with reduced and boiled one .Sometimes the oxidized and reduces forms run sligthly different. Buena suerte.
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