Can IP shift the MW of a protein? - (Feb/25/2008 )

I get pretty IP results, single band in the IP lane, and clean negative results. The problem is, the band is higher than I expected. I used two different antibodies (from the same species), including one used for IP, and they all give same results on the blotting. Could I trust the band? Can IP shift the MW of the protein?

I used a differnt beads this time, my last IP worked, the band is at right MW, but the negative control is not clean so I tried a differnt beads, and use protein G HRP as my secondary in the western blotting.

Please Please give me your advice.

Sparrow

I get pretty IP results, single band in the IP lane, and clean negative results. The problem is, the band is higher than I expected. I used two different antibodies (from the same species), including one used for IP, and they all give same results on the blotting. Could I trust the band? Can IP shift the MW of the protein?

I used a differnt beads this time, my last IP worked, the band is at right MW, but the negative control is not clean so I tried a differnt beads, and use protein G HRP as my secondary in the western blotting.

Please Please give me your advice.

Sparrow

Err, the protein is at 100 kD, the shift is at 120-140 kD? the gel resolve 30-100 kD best. let's say 120 kD.

Checking my blots again, I saw there is also a weak band at 120 kD in western blotting in addition to the strong 100kD band.But IP only have the 120 kD band. I have another antibody ( that previously worked for western very well, but not for IP. I stripped the IP blot (IP
with antibody A), blotting it again with antibody B, and detect the same 120 kD band, a bit less strong. But antibody B does not detect the 120kD band in western.

Sparrow[/quote]

how wide is the shift?
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