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Proteins Digest, Vol 22, Issue 15

Proteins Digest, Vol 22, Issue 15 - Protein Forum

Proteins Digest, Vol 22, Issue 15 - Protein Forum


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Old 03-31-2007, 10:13 PM
Dan Guire
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Default Proteins Digest, Vol 22, Issue 15



Hi All,

The kit is Pierce's CarboLink; I didn't really know if it's conventional to
mention those details here or not.

The gel itself doesn't bind the enzyme-linked test protein to a significant
extent. Until that is the gel has been exposed to antibody to the test
protein, whether the antibody's been pre-treated with Pierce's periodate
reagent or not. Strangely (in my opinion), pre-heating the antibody to 37C
works about as well as pre-treating it with the periodate.

I guess the antibody could be denatured by heating, made stickier to the gel
and yet still specific to its ligand, but that seems a stretch.

It's an excellent point, but an irrelevant antibody doesn't confer affinity
to the test protein (which is a mouse anti Rabbit~HRP conjugate).

I have to admit that I don't know how much protein antibody the gel is
taking up, because I haven't been measuring the content of what passes
through when that incubation is over. I have done a BCA on the gels from
the columns. The CarboLink gel itself is apparently reactive to the BCA
reagent, so it's hard to say how much protein is there.

I've tried Pierce tech support and been assured that the gel is passivated
enough, but I may try them again.

The other thing that occurs to me to try is tests with something like an
anti BSA so I can use BSA for the ligand, and see if any of these
preparations is allowing me to bind much of the test protein.

I've been trying pretty hard to make this work, and I appreciate your input;
have a good one

DanG



-----Original Message-----
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[mailtoroteins-bounces@oat.bio.indiana.edu] On Behalf Of
[Only registered users see links. ]
Sent: Saturday, March 31, 2007 12:01 PM
To: [Only registered users see links. ]
Subject: Proteins Digest, Vol 22, Issue 15

Send Proteins mailing list submissions to
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Today's Topics:

1. Re: affinity columns (Allison)
2. RE: RE: Re: affinity columns (Allison) (DK) (Dan Guire)


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

Message: 1
Date: Fri, 30 Mar 2007 16:40:06 -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:
to
reagent,
which
as
antibody

I haven't made my own affinity columns but some questions do come to mind:

Does your gel alone bind the test protein or does an Ab with a different
specificity also give the same effect? Because I agree with you that
enzyme-labelled proteins can sometimes be much too sensitive. Perhaps
the gel has some inherent "stickiness" and will bind a bit of Ab and a
bit of ligand without any treatment at all.

Does the kit come with any info as to how much antibody can be coupled
per gm of gel? If so, how does this compare with how much you can bind
without pretreatment.

What does the company that makes the kit say when you contact them?
Presumably they have lots of experience with troubleshooting and it is
in their best interests to help solve your problem for you.

Do you want to let us know the name of kit/company?

Allison


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

Message: 2
Date: Fri, 30 Mar 2007 17:35:06 -0500
From: "Dan Guire" <[Only registered users see links. ]>
Subject: [Protein-analysis] RE: RE: Re: affinity columns (Allison)
(DK)
To: <[Only registered users see links. ].indiana.edu>, <[Only registered users see links. ].indiana.edu>
Message-ID: <000301c7731b$aaf71820$4f6fa8c0@IsurTec.local>
Content-Type: text/plain; charset="us-ascii"

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-----
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[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
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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. ]

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When replying, please edit your Subject line so it is more specific
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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


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