Methods Digest, Vol 45, Issue 11

whyharish reddy via methods%40net.bio.net (by whyharish from gmail.com)
Wed Feb 18 19:47:08 EST 2009


Hi Thank you, for giving information on enzyme stability...!

On Mon, Feb 16, 2009 at 10:33 PM, <methods-request from oat.bio.indiana.edu>wrote:

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> Today's Topics:
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>   1. Re: STABILIZERS FOR ENZYMES (mainly XYLANASE)Methods Digest,
>      Vol 45, Issue 7 (Dr Engelbert Buxbaum)
>   2. Detergent concentration for nuclear membrane dissolving
>      (Werner Straube)
>   3. methylcellulose plating problem (Scott Brown)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Sun, 15 Feb 2009 08:39:38 -0400
> From: "Dr Engelbert Buxbaum" <engelbert_buxbaum from hotmail.com>
> Subject: Re: STABILIZERS FOR ENZYMES (mainly XYLANASE)Methods Digest,
>        Vol 45, Issue 7
> To: methods from net.bio.net
> Message-ID: <op.upd74c1k66vu6s from bengelbert-dm.rusm.rossu.loc>
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> Am 11.02.2009, 15:29 Uhr, schrieb whyharish reddy <whyharish from gmail.com>:
>
> > *I am unable to maintain my enzyme activity not more than 2 days, *
> > *please help in mainatining the activity of XYLANASE ENZYME for more than
> > 2-3 months, suggest some Enzyme Stablizers?*
>
> Have you done a literature search (scholar.google.com, PubMed)? Of hand I
> can help only with some general hints:
> - Stability is temperature dependent. As is generally known, enzymes can
> be    heat-denatured, but some are also cold-denatured.
> - Stability is strongly pH-dependent. Avoid storing enzymes near the pI,
> and at extreme pH values. Other than that, trial and error.
> - Some enzymes are sensitive to ionic strength and osmotic pressure. Keep
> both near physiological values. For intracellular enzymes use KCl, for
> extracellular NaCl to adjust ionic strenght. Manitol, sucrose, glycerol or
> similar polyols are used to maintain osmotic pressure.
> - Some enzymes require specific cofactors for activity and may be
> stabilised in their presence. Mg, Zn...
> - Sterile filter your preparation using a low-protein binding membrane
> (0.22 µm) and use aseptic techniques for handling. Add presevatives (Na
> azide, Thimerosal...) but check that they are compatible with the enzyme.
> - Check an inactive preparation for what actually happend, for example by
> SDS-PAGE, light scattering and CD-spectroscopy. Is the enzyme degraded
> (presence of proteolytic enzymes, microorganisms), forms aggregates or
> changes conformation? That info can help you develop more targeted
> approaches to the problem.
>
>
> ------------------------------
>
> Message: 2
> Date: Sun, 15 Feb 2009 21:29:53 +0100
> From: "Werner Straube" <straube from biochem.mpg.de>
> Subject: Detergent concentration for nuclear membrane dissolving
> To: <methods from magpie.bio.indiana.edu>
> Message-ID:
>        <36B75E885C4376419EC0020DA151B41C01149A1B from msx.w2k.biochem.mpg.de>
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>
> Hello everybody,
>
> A friend of mine is interested isolating nuclear proteins. After he has
> isolated the nuclei of cells he then wants to dissolve the nuclear
> membrane without destroying the interactions of nuclear proteins.
>
> We were already discussing the type and concentrations of detergents,
> however we did not get final clue.
>
> Can anyone help me out there with some suggestions or protocols or are
> there good references (protocols) in the literature (for general
> membrane dissolving and in particular nuclear membrane dissolving) ?
>
> Thank you very much,
>
> Werner
>
>
>
>
>
> ------------------------------
>
> Message: 3
> Date: Mon, 16 Feb 2009 12:23:26 +1100
> From: "Scott Brown" <SBrown from ccia.unsw.edu.au>
> Subject: methylcellulose plating problem
> To: <methods from magpie.bio.indiana.edu>
> Message-ID:
>        <2A67EA781EC7F949A2AB0A0D07A86C6A02972C3D from mail01.ccia.local>
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>
> Hi,
>
> This may be a long shot but i am banging my head against the wall over it.
> I have been doing some methocult plating, basically i take normal Nalm-6
> cells, add 1.6 mls methocult, 1.2 mls FCS, 1.2mls IMDM media and then i add
> dexamethosone at a conc. of 8uM.
> Last year this would kill the majority of the Nalm-6 cells (EC50 = 6nM),
> there were always some colonies that would grow through so i added some
> methotrexate to try and decrease the formation of colonies.
> For the past 6 months I have been getting equal numbers of colonies in my
> control (the nalms) as well as my experimental population of cells. I've
> checked my conc. of dex, repeated MTTs to confirm the EC50, repeated the
> plating several times but continue to get too many colonies being formed in
> the control. Previous plating showed a distinct difference in the amount of
> colonies formed in the control and the experimental situation.
>
> Does anyone have any ideas as to what may be going on or any suggestions
> for an experiment i may be able to do to get to the bottom of this?
>
> cheers
>
> Scott
>
> Scott Brown
> PhD Candidate
> Molecular Carcinogenesis Program
> Children's Cancer Institute of Australia for Medical Research
> High Street (PO Box 81)
> RANDWICK NSW 2031
> AUSTRALIA
>
> Phone: +61 2 9382 1829
> Fax: +61 2 9382 1850
> Email: sbrown from ccia.unsw.edu.au
> Web: www.ccia.org.au <http://www.ccia.org.au/>
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-- 
Y.HARISH KUMAR REDDY


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