Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protein Science > Protein Forum
Register Search Today's Posts Mark Forums Read

Protein Forum Protein Forum


RE: Re: affinity columns (Allison) (DK)

RE: Re: affinity columns (Allison) (DK) - Protein Forum

RE: Re: affinity columns (Allison) (DK) - Protein Forum


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 03-30-2007, 10:35 PM
Dan Guire
Guest
 
Posts: n/a
Default RE: Re: affinity columns (Allison) (DK)



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: [Only registered users see links. ]
[mailtoroteins-bounces@oat.bio.indiana.edu] On Behalf Of
[Only registered users see links. ]
Sent: Friday, March 30, 2007 12:02 PM
To: [Only registered users see links. ]
Subject: Proteins Digest, Vol 22, Issue 14

Send Proteins mailing list submissions to
[Only registered users see links. ]

To subscribe or unsubscribe via the World Wide Web, visit
[Only registered users see links. ]
or, via email, send a message with subject or body 'help' to
[Only registered users see links. ]

You can reach the person managing the list at
[Only registered users see links. ]

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" <[Only registered users see links. ]>
Subject: [Protein-analysis] Re: Enthalpy of protein
To: [Only registered users see links. ]
Message-ID: <1175183624.882753.238960@p15g2000hsd.googlegroups .com>
Content-Type: text/plain; charset="iso-8859-1"

On Mar 29, 1:20 pm, Frank K|ster <[Only registered users see links. ]> wrote:

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 <[Only registered users see links. ]>
Subject: [Protein-analysis] Re: Enthalpy of protein
To: [Only registered users see links. ]
Message-ID: <[Only registered users see links. ]>
Content-Type: text/plain; charset=us-ascii

"Protenger" <[Only registered users see links. ]> wrote:


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.


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.


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:
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" <[Only registered users see links. ]>
Subject: [Protein-analysis] RE: Re: affinity columns (Allison)
To: <[Only registered users see links. ].indiana.edu>
Message-ID: <000101c77230$e480ee40$4f6fa8c0@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: [Only registered users see links. ]
[mailtoroteins-bounces@oat.bio.indiana.edu] On Behalf Of
[Only registered users see links. ]
Sent: Thursday, March 29, 2007 12:01 PM
To: [Only registered users see links. ]
Subject: Proteins Digest, Vol 22, Issue 13

Send Proteins mailing list submissions to
[Only registered users see links. ]

To subscribe or unsubscribe via the World Wide Web, visit
[Only registered users see links. ]
or, via email, send a message with subject or body 'help' to
[Only registered users see links. ]

You can reach the person managing the list at
[Only registered users see links. ]

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 <[Only registered users see links. ]>
Subject: [Protein-analysis] Re: affinity columns
To: [Only registered users see links. ]
Message-ID: <[Only registered users see links. ]>
Content-Type: text/plain; charset=us-ascii; format=flowed

Dan Guire wrote:

conjugate
doesn't
a
you
use

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: [Only registered users see links. ] (DK)
Subject: [Protein-analysis] RE: Re: affinity columns (Allison)
To: [Only registered users see links. ]
Message-ID: <3C%Oh.2214$[Only registered users see links. ]>

In article <[Only registered users see links. ].net> , "Dan Guire"
<[Only registered users see links. ]> wrote:
which

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
[Only registered users see links. ]
[Only registered users see links. ]

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

Reply With Quote
Reply

Tags
affinity , allison , columns


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
antibody affinity chromatography question zhjwmy@yahoo.com Protein Forum 1 03-26-2009 12:57 PM
Source for Wizard/Merlin midiprep plastic columns? Jenny F. Protocols and Methods Forum 1 03-03-2008 07:53 AM
Proteins Digest, Vol 22, Issue 15 Dan Guire Protein Forum 0 03-31-2007 10:13 PM
Re: affinity columns (Allison) Dan Guire Protein Forum 2 03-30-2007 09:40 PM
ELISA HELP NEEDED Cant Get anything to Work (Allison) Dan Guire Protein Forum 1 03-28-2007 08:27 PM


All times are GMT. The time now is 01:37 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.14514 seconds with 16 queries