[ccp4bb] reductive alkylation / crystallization

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From: Kay Diederichs Kay {- dot -} Diederichs {- at -} UNI-KONSTANZ {- dot -} DE
Date: 2008-01-14

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From: Alexey Rak Alexey {- dot -} Rak {- at -} SANOFI-AVENTIS {- dot -} COM
Date: 2008-01-14

Subject: reductive alkylation / crystallization
From: Matt Vetting vetting {- at -} MEDUSA {- dot -} BIOC {- dot -} AECOM {- dot -} YU {- dot -} EDU
Date: 2008-01-14

I was interested in some of the finer points of the reductive methylation
procedure to dimethylate lysine residues for alternative crystallization
possiblilties.

The procedure is to incubate the protein with increasing concentrations
of formaldehyde and dimethylborane complex (as a reducing agent). The
protein concentration is usually in the 1-10 mg/ml range (0.1 to 1 mM)
protein (1 to 10 mM lysines), and the final formaldehyde and DMBC
concentrations are 80 and 50 mM. In some of the published procedures the
authors indicate that little formaldehyde will be remaining after the 20
hour reaction time.

So I was wondering what is the reason for disappearence of
formaldehyde. Does the DMAB reduce the aldehyde to an alcohol over time,
and does this occur rapidly or over a long time frame. What is the reason
for the extended overnight incubation period? Doesnt the reaction of
formaldehye with lysine occur rapidly? In general, why doesnt this
procedure lead to crosslinking (formaldehye is used as a fixitive agent in
many tissue preperations for EM)? Does the DMAB help keep the formaldehyde
in a monomeric form?

Thanks,
Matthew Vetting

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CCP4bb <-- 2008 <-- January 2008 <-- 14 January 2008

Previous message:
Subject: Re: raid array load question
From: Kay Diederichs Kay {- dot -} Diederichs {- at -} UNI-KONSTANZ {- dot -} DE
Date: 2008-01-14

Next message:
Subject: Re: crystallisation robot
From: Alexey Rak Alexey {- dot -} Rak {- at -} SANOFI-AVENTIS {- dot -} COM
Date: 2008-01-14