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How to dispose beta-Mercaptoethanol out of solutions

How to dispose beta-Mercaptoethanol out of solutions - Protocols and Methods Forum

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  #1  
Old 06-27-2006, 11:46 AM
Sebastian Gruenberg
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Default How to dispose beta-Mercaptoethanol out of solutions



Hi members,

I have an urgent question for the forum.

I'm doing KMnO4-footprinting experiments and unfortunately, BME
disturbes the KMnO4 reaction (it is also used to quench the reaction
later). Since there is BME in the buffers of the proteins used in my
reactions, I have to get rid of almost all the BME in the protein
solutions.

Does anybody now how to do this without damaging the sensitive
proteins?

Thank you very much.

Sebastian

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Fax.: ++49-941-943 2403

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  #2  
Old 06-27-2006, 05:34 PM
Austin P. So (Hae-Jin)
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Default How to dispose beta-Mercaptoethanol out of solutions

Sebastian Gruenberg wrote:

You could use a microdialyser with a molecular weight cutoff of about
1/5 the size of your protein. Alternatively, you can use a
microfiltration unit to buffer exchange (YM-3 up to YM-100 from Millipore)

Austin
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  #3  
Old 06-27-2006, 05:53 PM
Nick Theodorakis
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Default How to dispose beta-Mercaptoethanol out of solutions


Sebastian Gruenberg wrote:


Iodoacetic acid or (better yet) iodoacetamide will alkylate free
sulfydryls. Add a slight molar excess (e.g., 2-fold) over free
sulfhydryls and incubate in the dark for about 30 min. (Assuming this
will not affect the permanganate, which I don't know). Of course, if
your protein has free solvent-accessible -SH than they will be
alkylated as well.

Nick

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