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Membrane proteins


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6 replies to this topic

#1 VLNano

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Posted 09 September 2011 - 05:34 AM

Hi,

I am trying to isolate plasma membrane proteins from cells in vitro using streptavidin beads.
But I am having problems with immobilization...
Has someone had problems with this method?

Thanks.
Cheers

#2 mdfenko

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Posted 09 September 2011 - 07:20 AM

stupid question: did you biotinylate the plasma membrane proteins?
talent does what it can
genius does what it must
i do what i get paid to do

#3 VLNano

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Posted 09 September 2011 - 09:09 AM

Instead of biotinylate plasma membrane proteins, I immobilized concanavalin A (biotin conjugate) into the streptavidin beads for fishing my proteins.

#4 mdfenko

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Posted 09 September 2011 - 09:47 AM

we need to know the conditions.

concanavalin a will bind glucoside and mannoside moieties. there are other lectins which will bind other sugars.

not all membrane proteins are glycosylated (not all cytosolic proteins are not glycosylated). are you looking for a specific protein which you know is glycosylated?

by the way, you can purchase con a-agarose. no need to waste streptavidin-agarose.
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genius does what it must
i do what i get paid to do

#5 VLNano

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Posted 12 September 2011 - 01:11 AM

I want to capture membrane proteins glycosylated.
I am not using agarose, instead I am using strepatavidin magnetic beads.
So, I think that the problem will be in the bounding of ConA with the beads...

Thank you.

#6 VLNano

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Posted 14 September 2011 - 03:42 AM

Thank youmdfenko for your reply.
I think the quantity of ConA I had it was low...only next week i can repeat...
But you know any protocol to capture membran proteins using beads?

Thks

#7 mdfenko

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Posted 16 September 2011 - 11:07 AM

Thank you mdfenko for your reply.
I think the quantity of ConA I had it was low...only next week i can repeat...
But you know any protocol to capture membrane proteins using beads?
Thks

if you are looking for a specific protein then you can use immobilized antibodies
talent does what it can
genius does what it must
i do what i get paid to do




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