Same Non Specific Banding on Western using HA, FLAG and GFP detection - (Apr/07/2009 )
I have been running detections on a Western for a protein tagged with HA, FLAG and GFP.
I have seen the same strong non-specific banding pattern on all separate detections, using:
-monoclonal mouse-derived unconjugated anti-HA from another lab
-HRP conjugated anti-HA
-HRP conjugated anti-FLAG
-mouse-derived Unconjugated anti-GFP
I have tried more vigorous washing, which decreased the intensity of all the bands but removed none.
I have also diluted the antibodies to 1/20 000
Blocking has been done with 5% skim milk- I am going to try BSA next.
I am at a loss as to how to eliminate these non-specific bands- any ideas?
Is it common to get the same non-specific banding pattern using different antibodies?
Is it worth washing even more vigorously?
westerner on Apr 8 2009, 03:36 AM said:
I have seen the same strong non-specific banding pattern on all separate detections, using:
-monoclonal mouse-derived unconjugated anti-HA from another lab
-HRP conjugated anti-HA
-HRP conjugated anti-FLAG
-mouse-derived Unconjugated anti-GFP
I have tried more vigorous washing, which decreased the intensity of all the bands but removed none.
I have also diluted the antibodies to 1/20 000
Blocking has been done with 5% skim milk- I am going to try BSA next.
I am at a loss as to how to eliminate these non-specific bands- any ideas?
Is it common to get the same non-specific banding pattern using different antibodies?
Is it worth washing even more vigorously?
Hi there
I think the bands may just be degradation products of your protein in question. In the cell, little bits of the protein may get cleaved yet still retain the tag and therefore you will see multiple bands and not just the one of you full-length protein.
Chris
Are all the antibodies monoclonal mouse? Do you get the same band with just mouse IgG?
Also, why do you care if there is a non-specific band? Most antibodies result in several non-specific bands on Westerns. Is this band in an area you need to otherwise visualize?
The same bands with different antibodies could imply either of two things:
1) contamination with something in some of your solutions (probably the antibody solutions) that is detectable by your secondary.
2) you are using mouse antibody on mouse tissue or rabbit on rabbit tissue etc, anitbodies- try getting a primary raised in a different species.
Hi all,
all the unconjugated antibodies are monoclonal mouse- haven't tried polyclonal.
and yes, the protein i am trying to detect is expected to band at the same position as the non-specific banding. I have used an uninjected control sample (which does not contain the tagged protein) and it gives the same banding as the injected lanes, although there may be a slight difference in intensity not caused by differences in protein concentration.
it is possible there is antibody contamination, although i used primary antibody from another lab in one of the HA detections. i did use our lab's secondary antibody for that one, however the same result was given by the unconjugated and conjugated HA detections.
Also, the proteins are from zebrafish and i am using anti-mouse antibodies.
westerner on Apr 8 2009, 11:57 PM said:
all the unconjugated antibodies are monoclonal mouse- haven't tried polyclonal.
and yes, the protein i am trying to detect is expected to band at the same position as the non-specific banding. I have used an uninjected control sample (which does not contain the tagged protein) and it gives the same banding as the injected lanes, although there may be a slight difference in intensity not caused by differences in protein concentration.
it is possible there is antibody contamination, although i used primary antibody from another lab in one of the HA detections. i did use our lab's secondary antibody for that one, however the same result was given by the unconjugated and conjugated HA detections.
Also, the proteins are from zebrafish and i am using anti-mouse antibodies.
It is probably the result of the mouse IgG reacting to another protein. Use a polyclonal rabbit antibody instead.
cheers, I'll give it a go 
I haven't tried the polyclonal antibody yet, but I did try a HA detection using horse serum to block instead of skim milk, and the developed film showed up nothing. Has anyone had any experience with skim milk vs horse serum blocking?