[Protein-analysis] RE: RE: Re: affinity columns (Allison) (DK)

Dan Guire via proteins%40net.bio.net (by dguire from isurtec.com)
Fri Mar 30 17:35:06 EST 2007


Hi DK,

Yes it's a hydrazide-functional beaded agarose gel support, and the antibody
or other glycoprotein is oxidized with periodate to form reactive aldehydes.
I've conferred enough antibody activity to the gel without using the
periodate reagent to make me doubt that the principle of the kit's
preparation is sound.  Direct ELISAs are based on the stickiness of protein,
maybe the antibody just sticks to the agarose whether the reagent is used or
not.

DanG


-----Original Message-----
From: proteins-bounces from oat.bio.indiana.edu
[mailto:proteins-bounces from oat.bio.indiana.edu] On Behalf Of
proteins-request from oat.bio.indiana.edu
Sent: Friday, March 30, 2007 12:02 PM
To: proteins from magpie.bio.indiana.edu
Subject: Proteins Digest, Vol 22, Issue 14

Send Proteins mailing list submissions to
	proteins from net.bio.net

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

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

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


Today's Topics:

   1. Re: Enthalpy of protein (Protenger)
   2. Re: Enthalpy of protein (Frank K?ster)
   3. RE: Re: affinity columns (Allison) (Dan Guire)
   4. RE: Re: affinity columns (Allison) (DK)


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

Message: 1
Date: 29 Mar 2007 08:53:44 -0700
From: "Protenger" <yellowish from gmail.com>
Subject: [Protein-analysis] Re: Enthalpy of protein
To: proteins from net.bio.net
Message-ID: <1175183624.882753.238960 from p15g2000hsd.googlegroups.com>
Content-Type: text/plain; charset="iso-8859-1"

On Mar 29, 1:20 pm, Frank K|ster <f... from kuesterei.ch> wrote:
> "Protenger" <yellow... from gmail.com> wrote:
> > I ask my question in another format.
> > Suppose we have a native protein with its own intristic (absolute)
> > enthalpy, then by protein engineering we introduce a pair
> > of opposide charged residues on its surface so its stability
> > increases.
> > Does absolut (and not delta H) enthalpy of mutant decrease?
>
> What is absolute enthalpy, and what is it good for to know it?
>
> Regards, Frank
> --
> But this:> For fucks sake...
>
> is just offensive.  It should have an apostrophe(!)
> [MJ Ray, nowhere]

One of my fellows said "by introducing stabilizing interaction in
proteins (in its core or surface) its enthalpy will increase"
This sentence have confused me very much, because as I have learned
before reactions (for example protein folding) tend to
reduce their enthalpy by releasing heat so stabilized protein (native)
relative to intermediate one (for example molten globule state) must
have a lower enthalpy.
So what about engineered proteins that stabilized for example by
introduced salt bridges?
What is the deference between intrinsic (absolute) enthalpy between
them?
Or clearly, which of them has higher enthalpy?

Regards, Rahim




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

Message: 2
Date: Thu, 29 Mar 2007 18:59:57 +0200
From: Frank K?ster <frank from kuesterei.ch>
Subject: [Protein-analysis] Re: Enthalpy of protein
To: proteins from net.bio.net
Message-ID: <dt7td4-r58.ln1 from riesling.kuesterei.ch>
Content-Type: text/plain; charset=us-ascii

"Protenger" <yellowish from gmail.com> wrote:

> One of my fellows said "by introducing stabilizing interaction in
> proteins (in its core or surface) its enthalpy will increase"

That doesn't sound correct.  First of all, as has been pointed out, the
absolute enthalpy is both hard to get and quite boring.  The interesting
question, therefore: Which process is considered?  If it is complete
unfolding, then the quantity of interest is the enthalpy of
folding/unfolding.

> This sentence have confused me very much, because as I have learned
> before reactions (for example protein folding) tend to
> reduce their enthalpy by releasing heat so stabilized protein (native)
> relative to intermediate one (for example molten globule state) must
> have a lower enthalpy.

Err, are we talking about enthalpy or Gibbs (Free) enthalpy?

And what is it that confuses you?  Are you sure you were both talking
about the same process?  Naturally, if the (Gibbs) enthalpy of unfolding
decreases upon a mutation, the (Gibbs) enthalpy of folding will
increase.

> So what about engineered proteins that stabilized for example by
> introduced salt bridges?
> What is the deference between intrinsic (absolute) enthalpy between
> them?
> Or clearly, which of them has higher enthalpy?

Why are you interested in absolute enthalpies, when all that is
experimentally accessible is the change in enthalpy (or Gibbs enthalpy)
associated with a process?

Regards, Frank
-- 
But this:
> For fucks sake...
is just offensive.  It should have an apostrophe(!)
[MJ Ray, nowhere]


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

Message: 3
Date: Thu, 29 Mar 2007 13:34:31 -0500
From: "Dan Guire" <dguire from isurtec.com>
Subject: [Protein-analysis] RE: Re: affinity columns (Allison)
To: <proteins from oat.bio.indiana.edu>
Message-ID: <000101c77230$e480ee40$4f6fa8c0 from IsurTec.local>
Content-Type: text/plain;	charset="us-ascii"

Hi Allison,

I'm trying to prepare an immobilized-antibody affinity column.  I've tried
using a commercial kit that includes a gel which is intended to bind
antibodies that have been pretreated with a reagent that's included in the
kit.  My problem is that in the course of troubleshooting the procedure, I
found that just exposing the gel to the antibody solution without
pretreating the antibody with their reagent is enough to confer affinity to
the gel.  It binds my antigen about as well as if I had used their reagent,
so the antibody must be passively adsorbed there to a significant extent.

Columns prepared in this way can stand up to acid elutions and salt washes
about as well too.  So my question is:  Is it possible that it's not
preferable to covalently immobilize the affinity agent?  I doubt that, which
leads me to think that my method is misleading me.  

In testing the kit I've bought I've been using an enzyme-labeled protein as
ligand.  Since a small amount of enzyme can give back a large signal, the
total amount of protein is small.  Maybe I haven't been loading enough
ligand onto the columns to reflect the true amounts of immobilized antibody
on the differently-prepared columns.

Hey I might try doing a BCA on the gels themselves to see if there's more
protein on one than the other, or using a larger loading of an unlabeled
version of my ligand.  Any other thoughts will be much appreciated,

DanG



-----Original Message-----
From: proteins-bounces from oat.bio.indiana.edu
[mailto:proteins-bounces from oat.bio.indiana.edu] On Behalf Of
proteins-request from oat.bio.indiana.edu
Sent: Thursday, March 29, 2007 12:01 PM
To: proteins from magpie.bio.indiana.edu
Subject: Proteins Digest, Vol 22, Issue 13

Send Proteins mailing list submissions to
	proteins from net.bio.net

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

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

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


Today's Topics:

   1. Re: affinity columns (Allison)
   2. Re: Enthalpy of protein (Raoul Fleckman)
   3. Re: Enthalpy of protein (Frank K?ster)


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

Message: 1
Date: Wed, 28 Mar 2007 15:27:46 -0500
From: Allison <allison from nospam.com>
Subject: [Protein-analysis] Re: affinity columns
To: proteins from net.bio.net
Message-ID: <qeadnT45_8AqXJfbnZ2dnUVZ_sjinZ2d from mcgill.ca>
Content-Type: text/plain; charset=us-ascii; format=flowed

Dan Guire wrote:

> Hi Barbara,
> 
> The main point has already been made:  Your anti rabbit alphatase
conjugate
> can only detect the rabbit antibody down on the Maxisorp plate.  It
doesn't
> matter whether there is antigen for the capture antibody or not.  No doubt
a
> negative control, leaving out only the mouse lysate, would show you that. 
> 
> The series Allison outlined looks fine; the enzyme-conjugated anti mouse
> shouldn't bind unless there is mouse antibody bound to antigen there.  My
> point is that if you already have biotinylated anti ENAC of one species,
you
> can capture any antigen with the non-biotinylated anti ENAC of the other
> species, then detect with the biotin and a streptavidin-enzyme conjugate.
> And a negative control is a must :-)
> 
> DanG
> 
> P.S. Do you have any affinity column experience by any chance?  I could
use
> some help in that arena...
> 
>

I have done a bit....what is your question?  If I can't help I am sure 
someone else can.
Allison


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




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

Message: 4
Date: Fri, 30 Mar 2007 03:37:34 GMT
From: dk from no.email.thankstospam.net (DK)
Subject: [Protein-analysis] RE: Re: affinity columns (Allison)
To: proteins from net.bio.net
Message-ID: <3C%Oh.2214$qe1.1608 from newsfe04.lga>

In article <mailman.563.1175200089.5139.proteins from net.bio.net>, "Dan Guire"
<dguire from isurtec.com> wrote:
>Hi Allison,
>
>I'm trying to prepare an immobilized-antibody affinity column.  I've tried
>using a commercial kit that includes a gel which is intended to bind
>antibodies that have been pretreated with a reagent that's included in the
>kit.  My problem is that in the course of troubleshooting the procedure, I
>found that just exposing the gel to the antibody solution without
>pretreating the antibody with their reagent is enough to confer affinity to
>the gel.  It binds my antigen about as well as if I had used their reagent,
>so the antibody must be passively adsorbed there to a significant extent.
>
>Columns prepared in this way can stand up to acid elutions and salt washes
>about as well too.  So my question is:  Is it possible that it's not
>preferable to covalently immobilize the affinity agent?  I doubt that,
which
>leads me to think that my method is misleading me.  

Knowing what is in the kit anf how it works can do wonders to 
understanding what's going on and troubleshooting. Do they tell you the 
principle and the chemistry involved? 

DK


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

_______________________________________________
Proteins mailing list
Proteins from net.bio.net
http://www.bio.net/biomail/listinfo/proteins

End of Proteins Digest, Vol 22, Issue 14
****************************************



More information about the Proteins mailing list