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I've just read an article Rab10 GTPase regulates ER dynamics and morphology - Nature Cell Biology 15, 169–178 (2013) doi:10.1038/ncb2647. In this paper, to identify Rab proteins in ER, first they isolated ER vesicles. Then washed them to remove cytosolic proteins. After washing, these vesicles were solubilized by detergent. Then, it was loaded on to a GTP-agarose column (to bind GTP binding proteins to the column). In the last step GTP binding proteins were eluted with GTP and analysed by SDS-PAGE.

As a control (before loading on to the column) they used GTP gamma S, because it prevents GTP-binding proteins from binding onto the column. When I looked at their figure; on the control lane, there are lots of bands! So I'm confused :(If it prevents GTP-binding proteins from binding onto the column, after eluting from GTP agarose column why there are bands? And what can these bands be?)

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If you can add a link to the article (hopefully it's open-access) that would be helpful in interpreting the results. –  MattDMo Feb 13 '13 at 22:53
    
The title of this article is ''Rab10 GTPase regulates ER dynamics and morphology'' Link: nature.com/ncb/journal/v15/n2/full/ncb2647.html Unfortunately, it is not open-access.. –  Armacino Feb 16 '13 at 20:36
    
I can still see the figures, though. Which one did you have the question about? –  MattDMo Feb 16 '13 at 22:03
    
Figure 1 d. Thank you for your help. –  Armacino Feb 17 '13 at 1:29
    
Sort of related: biology.stackexchange.com/questions/3258/… –  nico Feb 17 '13 at 8:23
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1 Answer

up vote 5 down vote accepted

This is the figure the question is about. On the right is the control experiment with GTP-γS, on the left without it:

enter image description here

The bands that are visible in both experiments are unspecific binding. If GTP-γS doesn't affect their presence, the mechanism by which they bind to the column can't be specific to the GTPase functionality.

The proteins the authors were after are those that are present in the experiment without GTP-γS, but are not eluted in the experiment with GTP-γS. Those are binding via an GTPase functionality to the GTP on the column. They are marked with arrow heads in the figure.

The general idea behind this control experiment is to distinguish specific binding from unspecific binding to the column. Being able to do that means that the authors had fewer proteins they needed to investigate further, saving them some work.

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Thank you so much! ;) –  Armacino Feb 17 '13 at 20:10
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