I ran 12% SDS PAGE using tris glycine buffer. after transfer i cut two lanes for ubiquitin detection and 4 lanes for beta actin. after development i got a strong intense single band for b actin. However for ubiquitin (8.5kDa) (polyclonal) i got one intense band and a few light bands. Problem is that the intense band is exactly at the same position as beta actin (46kDa). Now I am in doubt if the band was even specific for beta actin or was it some non specific band. What could be the reason for band at same position for two different proteins with very diferent mol weights.
Didnt you load a molecular marker/ladder in the gel. You could compare the size of the band and than make a decision which one of the two is nonspecific..
antibody for beta actin is monoclonal and ubiquitin is polyclonal. i didnt use marker.. So using only marker we can specify the band position?
Yes, you need to run a pre-stained protein marker in addition to your samples everytime because via the marker bands you can also decide that your gel was not running to long but long enough to get your proteins of interest separated. In your case to detect ubiquitin which is roughly 9 kDa big (in addition to beta actin), I would recommend a 15% or 18% SDS polyacryamide gel anyway. Good luck!
yes i m trying 15% gel for ubiquitin now.. thanks a lot for ur suggestion
it will seem to all of you very stupid but it happens ...
have you washed the stripes, or incubated them with the 2nd ab in a same container?
Abs, when are in large excess , pass from a filter to another...
No incubation was done in different containers.. However sencondary for both of them is same
then during the 2nd antibody incubation (of 1h, I suppose)
the anti- actin antibodies bound to their target with only one binding-site can bind to the other filter through the second binding-site
think about
it is the only possibility...to explain your result
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