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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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Western blot with HA-tagged protein expression in K562
Posted by: Ivy (IP Hidden, Unregistered user, )
Date: April 8, 2005 09:27PM
Hi,
I tried to express some HA-tagged proteins in K562 cells many times, 4 different proteins actually... but whenever I tried to do a Western blot with a rabbit polyclonal HA antibody that's known to work (at least for the same proteins expressed in E. coli cells), I could never detect the HA-tagged protein bands. I have tried two different vector backbones with the 4 proteins, different lysis protocols.... and still could not get the Western to work. Does anyone have any idea? Thanks a lot! ivy
Re: Western blot with HA-tagged protein expression in K562
Posted by: ECLman (IP Hidden, Unregistered user, )
Date: April 8, 2005 10:46PM
(1) Transfection problem??
(2) Toxic proteins?? (3) Try monoclaonal anti-HA, protein expressed in coli is different from mammalian cells.
Re: Western blot with HA-tagged protein expression in K562
Posted by: Ivy (IP Hidden, Unregistered user, )
Date: April 9, 2005 07:16AM
I think the transfection was ok because my cells eventually become geneticin-resistant (from the plasmid I put in). I'm not sure if the proteins are toxic or will be immediately degraded by the cells. I've tried monoclonal anti-HA Ab and didn't make a difference. The rabbit polyclonal HA ab I used can recognize another HA-tagged protein in HeLa cells. So I'm not sure what my problem is....
Re: Western blot with HA-tagged protein expression in K562
Posted by: ECLman (IP Hidden, Unregistered user, )
Date: April 10, 2005 02:46PM
Do
(1) a transient transfection of k562 and see if you can detect HA-tagged proteins; or (2) in vitro transcription/translation and Western using HA antibody to confirm your sequence is right in-frame and can be expressed??!!
Re: Western blot with HA-tagged protein expression in K562
Posted by: Ivy (IP Hidden, Unregistered user, )
Date: April 11, 2005 05:44PM
good idea. i should do the in vitro transcription/translation first. thanks!
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