I have a urgent questuion,

Sayyari, Mohammad via methods%40net.bio.net (by msayyar from gwdg.de)
Mon Oct 30 04:42:23 EST 2006


Dear Sir/Madam

I am trying to measure oxidizer -reducer microorganism in rhizosphere soil.
Can one tell me the method? I would appreciate it, if you could help
me.

Best regards,
Mohammad



________________________________

From: methods-bounces from oat.bio.indiana.edu on behalf of
methods-request from oat.bio.indiana.edu
Sent: Wed 10/25/2006 7:01 PM
To: methods from magpie.bio.indiana.edu
Subject: Methods Digest, Vol 17, Issue 24



Send Methods mailing list submissions to
        methods from net.bio.net

To subscribe or unsubscribe via the World Wide Web, visit
        http://www.bio.net/biomail/listinfo/methods
or, via email, send a message with subject or body 'help' to
        methods-request from net.bio.net

You can reach the person managing the list at
        methods-owner from net.bio.net

When replying, please edit your Subject line so it is more specific
than "Re: Contents of Methods digest..."


Today's Topics:

   1. Re: Wessel-Flugge Method for Protein Precipitation (DK)
   2. RE: Methods Digest, Vol 17, Issue 23 (Sayyari, Mohammad)


----------------------------------------------------------------------

Message: 1
Date: Wed, 25 Oct 2006 02:06:28 GMT
From: dk from no.email.thankstospam.net (DK)
Subject: Re: Wessel-Flugge Method for Protein Precipitation
To: methods from net.bio.net
Message-ID: <DEz%g.56$Zj7.30 from newsfe03.lga>

In article <mailman.189.1161698277.23274.methods from net.bio.net>, Jeremy Kamil
<jeremy.kamil from gmail.com> wrote:
>Hi, I'm trying to concentrate dilute protein mixtures in my tandem 
>affinity purification eluates for mass spec analysis. Specifically, I 
>need to get rid of the EDTA and EGTA as well as the 0.1% tween or 
>NP-40 that is in my elution buffer.
>
>I saw that some people use the Wessel-Flugge method (Anal Biochem 
>1974) for this application (Methanol, Chloroform precipitation). 
>However, in my hands I found it didn't seem to work great, at least I 
>don't see a nice pellet.
>
>I've had some success in the past with TCA but often end up with some 
>residual acidity even after many acetone washes. Anyway, at least 
>with TCA I got a nice pellet every time.
>
>My question re: Wessel-Flugge is, HOW MUCH OF THE UPPER LAYER ARE YOU 
>SUPPOSED TO REMOVE??? Should I get rid of the upper phase all the way 
>down to the chloroform layer, or leave a bit above of the chloroform??
>
>The original publication says to remove the upper layer above the 
>chloroform and interphase. But I don't see much of an interphase, 
>except in some samples where I saw a diffuse band of white above the 
>chloroform layer on the bottom. so, in case that diffuse white stuff 
>was my protein, I left maybe 150 uL on top of the chloroform. In the 
>end I did not see much, if any, precipitate and was pretty 
>disappointed since the method seemed so promising.
>
>any advice would be very much appreciated. I found the description by 
>Wessel and Flugge pretty cursory.

I don't know, it always worked extremely well for me. Beats TCA any
day. I always remove the aqueous layer as completely as I can -
basically, until the meniscus collapses. I also always spin at all steps
much harder than, IIRC, indicated (1 min at 12K). The protein is on
interphase, so it is not a good idea to remove it. Don't think it's
detergents causing it in your case, since I used to remove 1%
Triton X-100 with no problems. Maybe try using more chloroform
during the initial denaturing step?

DK



------------------------------

Message: 2
Date: Wed, 25 Oct 2006 11:11:58 +0200
From: "Sayyari, Mohammad" <msayyar from gwdg.de>
Subject: RE: Methods Digest, Vol 17, Issue 23
To: <methods from oat.bio.indiana.edu>
Message-ID:
        <496AACD85991444AA5AFABD263EE3FAB9DAE85 from VS2.exc.top.gwdg.de>
Content-Type: text/plain;       charset="iso-8859-1"

Dear Sir/Madam

I am trying to measure oxidizer -reducer microorganism in rhizosphere soil.
Can one help me and tell the method? I would appreciate it, if you could help
me.

Best regards,
Mohammad

  _____ 

From: methods-bounces from oat.bio.indiana.edu on behalf of
methods-request from oat.bio.indiana.edu
Sent: Tue 10/24/2006 7:02 PM
To: methods from magpie.bio.indiana.edu
Subject: Methods Digest, Vol 17, Issue 23



Send Methods mailing list submissions to
        methods from net.bio.net

To subscribe or unsubscribe via the World Wide Web, visit
        http://www.bio.net/biomail/listinfo/methods
or, via email, send a message with subject or body 'help' to
        methods-request from net.bio.net

You can reach the person managing the list at
        methods-owner from net.bio.net

When replying, please edit your Subject line so it is more specific
than "Re: Contents of Methods digest..."


Today's Topics:

   1. Re: low molarity borate and acetate agarose gels (Pow Joshi)
   2. Re: Stripping Solution (Pow Joshi)
   3. Wessel-Flugge Method for Protein Precipitation (Jeremy Kamil)
   4. pEGAD,pGreenII and p35S-GFP-JFH1 (Jinxiang Wang)


----------------------------------------------------------------------

Message: 1
Date: Mon, 23 Oct 2006 15:41:49 -0500
From: "Pow Joshi" <pow.joshi from gmail.com>
Subject: Re: low molarity borate and acetate agarose gels
To: "Ho-Leung Ng" <holeung from berkeley.edu>
Cc: Methods from magpie.bio.indiana.edu
Message-ID:
        <710764ea0610231341g7406872egc17c77adbc37d2a4 from mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed

Hi,

frankly, I had run, regularly, some TBE gels (tris borate EDTA) and
the protocol is available in the Molecular cloning book, especially
the old editions by Tom Maniatis.....
You could use 0.5X instead of 1x buffer; alternatively, you could use
low voltages.
I used to find the resolutions comparable .... although it's easy to
run TAE gels if you wish to isolate/ purify your DNA.

hope this helps

pow
On 10/19/06, Ho-Leung Ng <holeung from berkeley.edu> wrote:
>      What are people's experiences using the Brody and Kern (Biotechniques
> 36:214 and 37:598) low molarity sodium borate and lithium acetate
> agarose gels? They do run very fast with little heat, but we often
> get very smeared bands, with borate performing better than acetate.
> There is a lot of band quality variability from run to run. Any tips
> would be appreciated!
>
>
> ho
> UC Berkeley
>
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>



------------------------------

Message: 2
Date: Mon, 23 Oct 2006 16:20:20 -0500
From: "Pow Joshi" <pow.joshi from gmail.com>
Subject: Re: Stripping Solution
To: "Christian Praetorius" <prae from gmx.net>
Cc: Methods from magpie.bio.indiana.edu
Message-ID:
        <710764ea0610231420i24c17122y2e75e60b1e9674f0 from mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed

On 10/15/06, Christian Praetorius <prae from gmx.net> wrote:
> Maryam <mzargh from yahoo.ca> wrote:
>
> >Does anybody know if NaOH solution can be used as a stripping solution? if
> So, what should be the final concentration?

you might want to add some beta mercaptoethanol or DTT as well ...
this would cleave the -S-S- linkages in the antibodies (again assuming
you want to strip western blots)

pow
>
> What do you want to strip?
>
> Christian
>
> --
> X-no-Sig: yes
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>



------------------------------

Message: 3
Date: Tue, 24 Oct 2006 02:29:19 -0400
From: Jeremy Kamil <jeremy.kamil from gmail.com>
Subject: Wessel-Flugge Method for Protein Precipitation
To: methods from magpie.bio.indiana.edu
Message-ID: <E4DEA952-3A7A-45B8-B8D8-F9AF2114DC17 from gmail.com>
Content-Type: text/plain; charset=US-ASCII; delsp=yes; format=flowed

Hi, I'm trying to concentrate dilute protein mixtures in my tandem
affinity purification eluates for mass spec analysis. Specifically, I
need to get rid of the EDTA and EGTA as well as the 0.1% tween or
NP-40 that is in my elution buffer.

I saw that some people use the Wessel-Flugge method (Anal Biochem
1974) for this application (Methanol, Chloroform precipitation).
However, in my hands I found it didn't seem to work great, at least I
don't see a nice pellet.

I've had some success in the past with TCA but often end up with some
residual acidity even after many acetone washes. Anyway, at least
with TCA I got a nice pellet every time.

My question re: Wessel-Flugge is, HOW MUCH OF THE UPPER LAYER ARE YOU
SUPPOSED TO REMOVE??? Should I get rid of the upper phase all the way
down to the chloroform layer, or leave a bit above of the chloroform??

The original publication says to remove the upper layer above the
chloroform and interphase. But I don't see much of an interphase,
except in some samples where I saw a diffuse band of white above the
chloroform layer on the bottom. so, in case that diffuse white stuff
was my protein, I left maybe 150 uL on top of the chloroform. In the
end I did not see much, if any, precipitate and was pretty
disappointed since the method seemed so promising.

any advice would be very much appreciated. I found the description by
Wessel and Flugge pretty cursory.

thanks,

Jeremy





------------------------------

Message: 4
Date: Tue, 24 Oct 2006 19:39:49 +0800
From: Jinxiang Wang <jinxwang from scau.edu.cn>
Subject: pEGAD,pGreenII and p35S-GFP-JFH1
To: arab-gen from magpie.bio.indiana.edu, methods from magpie.bio.indiana.edu
Message-ID: <453DFB85.40303 from scau.edu.cn>
Content-Type: text/plain; charset=GB2312

Hello,All,

I appreciate someone who would like to share pEGAD,pGreenII and
p35S-GFP-JFH1 with me to construct GFP-tagged plasmid to transform
/Arabidopsis/. Thank you in advance.

Jinxiang

--
Jinxiang Wang Ph.D
College of Resources and Environment
Root Biology Center
South China Agricultural Univeristy
Guangzhou 510642, P.R.China
Tel:0086-20-85280156
Fax:0086-20-85281829
Email:jinxwang from scau.edu.cn



------------------------------

_______________________________________________
Methods mailing list
Methods from net.bio.net
http://www.bio.net/biomail/listinfo/methods

End of Methods Digest, Vol 17, Issue 23
***************************************





------------------------------

_______________________________________________
Methods mailing list
Methods from net.bio.net
http://www.bio.net/biomail/listinfo/methods

End of Methods Digest, Vol 17, Issue 24
***************************************





More information about the Methods mailing list