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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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Help to a student
Posted by: mobio (IP Hidden, New member, 3)
Date: April 24, 2005 02:11PM
Hello,
I should write some kind of proposal and I need to ask one question. I have a small peptide (neuropeptide) of known structure for which receptor is unknown. Model organism is C. elegans. My assumption is that receptor is GPCR... So I would be extremely grateful to a person who can give me and reference (to an article or a book) with overview of the biochemical techniques that I can use to discover the receptor. If someone could give me some hint about techniques that would be great too. Just notice that I am looking for biochemical protein techniques (not RNAi for example). Thank you in advance! Best I.
Re: Help to a student
Posted by: 5'-ATCG (IP Hidden, Unregistered user, )
Date: April 24, 2005 03:00PM
1. Try to label your peptide;
2. Use this tagged peptide to fish out your putative receptor; 3. Clone out this receptor, characterize it in vitro and in vivo..... Search the required info on internet and literature is also a good training for a student. Direction is given, try to search for yourself. Believe me, a review article or a book won't be enough (to cover every aspect).
Re: Help to a student
Posted by: mobio (IP Hidden, New member, 3)
Date: April 24, 2005 06:01PM
Thank you very much for the help.
I had same idea and I was searching the net for couple of days but I couldn't find suitable article on this so I decided to try forum to short my time... I think review is good starting point since I will be able to look for the references. Thank you anyway for your time Best I
Re: Help to a student
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: April 25, 2005 01:02PM
Hello,
What 5'-ATCG have suggested is the the best strategy for acpturing your receprtor, where you can biotin label your peptide (deos it have a cystine in it?) and then incubate your biotin labeled peptide in the presence of a homobifunctional witha sulhydryl reactive group, water soulble and membrane impermeable cross linker . these conditions will aloow your peptide to cross link to the receptor and then you can run your cell lysate over a streptavidin column where your peptide will bind as complex with the targeted receptor. In order to release the target receptor from the peptide include a reducing agent (DTT or Mercaptoethanol) in your elution buffer to reverse the cross linking and you can collect the receptor in the elution buffer and then run it on SDS-PAGE and then perform Far Western analysis to further confirm that your peptide specifically binds to this receptor. If all goes well and you get a detectable band by either Comassie or Silver stain send it for MS analysis to obtain the identity of this receptor. There are several examples in the litrature for similar approaches but you need to look at each appraoch individually and check Pierce website for further information on cross linkers and biotinylation reagents and reaction conditions. Good Luck!
Re: Help to a student
Posted by: mobio (IP Hidden, New member, 3)
Date: April 25, 2005 03:40PM
Wow thanks bassamfahmawi it was really helpful.
I spent lot of time searching and I think runing cell lysat over column will not be so helpful because my receptor is most probably GPCR. As you know most GPCRs are functional and able to bind to their activating endogenous ligands only when embedded in the appropriate membrane environment. But cross linking is great idea. Maybe I can crosslink it on the cell membrane and than purify membrane fraction and search for the complex? I don't have technical limitations, I just need good idea. Thank you for your time. Best I Edited 1 times. Last edit at 04/25/05 03:46PM by mobio.
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