Difference in mobility of IP Importin Beta and whole cell lysate ImportinB

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scchaudhary
scchaudhary's picture
Difference in mobility of IP Importin Beta and whole cell lysate ImportinB

Dear friends!!
I am confused with a small experimental result.
I got there is different in mobility of immunoprecipitaed importin Beta with that of importin ateBeta from whole cell
lysate. i did ip with gfp ab. from GFP-fusion construct  containing NLS motif transfected cell sample  and their whole cell lysate  followed by WB with Importin Beta. I found IP  importin Beta detected at a higher shift than from whole cell lysate and I became confused.
Can you suggest me why it is happening so?
every time I got same results.
so, what would be the reseason for difference in mobility?
If you help me I will be grateful to you.

Thank you.

shubhash chaudhary,

Chin Fen Teo
Chin Fen Teo's picture
Hi shubhash chaudhary,

Hi shubhash chaudhary,

How much is the mass shift? Does it correspond to the Mw of GFP? What kind of tranfection efficiency you are getting?
If the shift matches GFP Mw and the transfection efficiency isn't that high, an obvious explanation is that, the WB on whole cell lysate is picking up endogenous form of Importin b.

Good luck.