Bands in the wrong place

[jasmith456]

I'm trying to western blot stathmin in cell lysate from human cancer cells, but my bands are too high. Stathmin is supposed to appear at 19 kDa, but I only get a single band at 35 kDa. When I run purified stathmin, the band is too low! It's around 10 kDa. Any idea what could be happening? There should be stathmin in my cell lysates, so even if the antibody is binding another protein, there SHOULD be two bands.
Maybe it's my gel? I'm using a pre-made Lonza 12% gel.
Has anyone had an experience like this?
Thanks!

[Anastasia_S]

Do you have protease inhibitors in your lysis buffer? Upon death, cellular proteases will chop up material if not inhibited, so this may cause the low MW band.

Does your protein have a glycosylated form (and hence higher MW)?

Are you sure in your MW ladder? The stuff I use from Fermentas seems to have changed over the years, so I have to watch which batch I'm using (like the stuff on the website is different from the batch that arrived not long ago and is also different from the 2007 stuff I had before)

[moganti]

Hello everyone,

I have been doing the Western blot since 3 months.I have loaded 30ug of samples and first checked with alpha tubulin and then added the desired antibody (cleaved caspase-3) .....I got the signals with controls but not with the antibody that I want.....Can anyone please give me the solution to get the results....
becoz I didnt find my signals .....

I added 5%milk/TBS-T
Primary antibody is cleaved caspase-3(1:1000)
Secpndary antibody is antirabbit (1:10,000)