Proposal for strategy to determine 2 protein interactions? - suggest sugges! (May/26/2005 )

any good ideas how can i suggest with items below?

Complementary DNA of Protein A and B
Two mice
A histadine-tag plasmid and a gst tag plasmid
E.Coli bacteria for producing recombinant proteins
Ni-NTA beads and glutathione beads
Anti-immunoglobulin beads
Reagents/materials for SDS PAGE and western blotting
A surface plasmon resonance machine

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Try an IP with one antibody after crosslinking and detect with the other

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you can also do a far western
first you separate total proteins in a classical page gel, tranfert them on membrane. Do a process of denaturation renaturation of proteins on the membrane. you reveal positions of your both proteins by antibody...

Then you radiolabel one of the proteins of interest and incubate with your membrane and gonna see if interaction occurs or not.

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Yeast two hybrid
GST-pull down
IP
co-localization in cells

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Ex.

His-tagged Protein A
GST-tagged protein B
(got from E coli expression sys)

Use an Ni-NTA flowchip in your SPR machine.

Immobilize His-tagged protein A onto the flowchip
Inject protein B over the chip, you will see interaction if any

Dont forget to include control experiments

Hope this is not foolish suggestion!

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To determine 2 protein interaction - yeast two hybrid system

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do you mean
-screen bait (proteinA) against a whole library and hope your expected protein (proteinB) will be picked up
or
- just screen one bait (proteinA) against one prey (proteinB) and look for activation of the reportergenes
?

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Is the latter a good strategy? Can you use the Y2H strategy in the following way : screening the bait against just one AD-protein and not against a whole library?

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Yes you can use the Y2H to scan one bait and one pray. And to confirm it further you can even change the bait and pray (i mean interchanging bait and pray).
Hope this will help you!

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Is this a part of ur homework?

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