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Tagged recombinant protein has different mobility? - (Nov/28/2012 )

I did an expression of Adh gene in BL21 (DE3). I purified the protein using histidine tag column. Then, I run the total protein and also purified Adh protein in same SDS PAGE gel. BUT, the purified histidine tagged protein has a quite larger size than expected size. anyone can explain why this happen?Is it tagged recombinant protein has different mobility? anyone can explain this to me?

-An-nur Al-Irsyad-

It is relatively common for proteins not to migrate at their expected sizes. Adding a tag could easily alter the charge on the protein, and thereby alter how it runs. SDS PAGE gels are not totally based on protein size, with the SDS creating the all of the charge; PI and a few other factors also play a role.

-bob1-

SDS is detergent that give negative charge on protein-correct me if im wrong..
I though only native page rely on the protein charge, pI

-An-nur Al-Irsyad-

An-nur Al-Irsyad on Wed Jan 23 06:12:44 2013 said:


SDS is detergent that give negative charge on protein-correct me if im wrong..
I though only native page rely on the protein charge, pI
What you say is true. Native PAGE does only rely on charge and size, but SDS-PAGE also has some component of charge migration, despite the SDS.

-bob1-

this may give you some insight:
Attached File

-mdfenko-

this may give you some insight:

I find that document rather confusing and not very clear.

They should have added all the pictures of the gels ran. 

They calculate a value of 37kda (non linear), but do not show the pictures of those gels. The only one that is shown , shows a band that is clearly below the 37kda band and has a rather perfect curve of the standards. I would have loved to see where the band was actually on the other gels because a calculated value of 37kda and then a band that is clearly below, does rings some alarm bells.

I am not sure you understand my point? 

To me it is a weird document. 

-pito-

it's been a long time since i read that paper but, if memory serves me, i believe they calculated the mw by its sequence, not by its mobility in another gel.

-mdfenko-