Is my antibody specific? - (Jul/01/2008 )
Hello everybody!
I am trying to see if the new antibody I received is specific for "my" protein. I have run a western blot with a E.coli-lysate and 2 different antibody quantities (1 and 0.2 ug). I alway run a control with just E. coli.
In the lane with the sample, there is a big double band at the right size, that should be my protein (even if the band shouldn't be double...). I tested it with an anti-his antibody directed against the his-tag of the expressed protein.
What are the other bands? Would you say that the antibody is specific enough?
Thank you very much![attachment=4919:antibody_specifity.jpg]
I am trying to see if the new antibody I received is specific for "my" protein. I have run a western blot with a E.coli-lysate and 2 different antibody quantities (1 and 0.2 ug). I alway run a control with just E. coli.
In the lane with the sample, there is a big double band at the right size, that should be my protein (even if the band shouldn't be double...). I tested it with an anti-his antibody directed against the his-tag of the expressed protein.
What are the other bands? Would you say that the antibody is specific enough?
Thank you very much![attachment=4919:antibody_specifity.jpg]
I am confussed. did you get a single band when you try with anti-his antibody. If yes, then the two bands could be your desire protein. one you trasformed cntaining extra marker sequence increasing your protein weight. the other could be the endogenous protein present in E. coli. If I am correct then your antibody is specific.
I am trying to see if the new antibody I received is specific for "my" protein. I have run a western blot with a E.coli-lysate and 2 different antibody quantities (1 and 0.2 ug). I alway run a control with just E. coli.
In the lane with the sample, there is a big double band at the right size, that should be my protein (even if the band shouldn't be double...). I tested it with an anti-his antibody directed against the his-tag of the expressed protein.
What are the other bands? Would you say that the antibody is specific enough?
Thank you very much![attachment=4919:antibody_specifity.jpg]
I had encountered similar situation when expression his-tagged protein. Could it be a degradation product (the his-tag of the protein is degarded, resulting a minute size shift) or modification of your protein? (e.g phosphorylation?). I think your control is really good, is it cell lysate?
Thank you everybody for the answers!
I have come to the same conclusion...degradation of the protein. Or maybe the double band comes having accidentally 2 transcription starts, I have to check this.
And yes, it's all pure cell lysate.