[Histonet] RE: Differences between WB antibody and IHC antibody

[JR R]

When I'm trying a new antibody for IHC, I will typically test several concentrations of the antibody and ALSO several different antigen retrieval methods side-by side.  I typically do Heat Induced Epitope retrieval at pH 6.0 and 9.0, and also trypsin digestion for 30 minutes.

Jerry Ricks
Research Scientist
University of Washington
Department of Pathology

> Date: Fri, 2 Jan 2009 17:14:12 -0500
> From: anh2006 <@t> med.cornell.edu
> To: talulahgosh <@t> gmail.com; histonet <@t> lists.utsouthwestern.edu; tifei <@t> foxmail.com
> Subject: Re: [Histonet] Differences between WB antibody and IHC antibody
> CC: 
> 
> Whether or not an antibody can be used for western vs IHC depends on 
> many factors.
> 
> Some antibodies can be used for both, some can be only used for one 
> vs another, and some cannot be used for either.
> 
> There are quite a few factors to consider and here are a few from the 
> top of my head (and I am hoping people can add to this list):
> 
> (1) Epitope recognition
> 
> Western blotting often times (but not always of course) involves the 
> denaturation of proteins as well as breaking of disulfide bonds etc. 
> Samples are often boiled in denaturing agents such as 
> beta-mercaptoethanol or DTT and are run in SDS containing buffers and 
> gels. In doing this certain epitopes may be revealed enabling a 
> specific antibody to work on western vs IHC. Along those same lines, 
> this denaturation of proteins can also destroy epitopes, because 
> proteins are no longer in their native conformations, disabling the 
> efficacy of certain antibodies.
> 
> Similarly, IHC involves fixation, processing, sectioning, heat 
> retrieval etc. Fixation alone can alter epitopes dramatically thereby 
> precventing a certain antibody from binding. On the contrary, 
> proteins are often well preserved in a native conformation in IHC 
> (especially in fresh frozen sections).
> 
> (2) Access
> 
> Because western blotting involves protein lysates of cells which have 
> been busted open by a variety of methods, all of the proteins in a 
> cell should be accessible to the antibody. In IHC on sections or on 
> coverslips etc, the cell structures are largely preserved intact. 
> Therefore certain cell compartments may need further processing to 
> grant access of the big bulky antibody to the inside of the 
> compartments. Hence part of the reason why we do "antigen retrieval" 
> with enzymes and detergents etc. Similarly because in western, one 
> might spin out fractions before running on a gel if you are searching 
> for a nuclear protein or other protein localized to a certain 
> compartment you must be sure you actually have the compartment 
> preserved in a western prep where you always would have it present in 
> an intact IHC section.
> 
> (3) Sensitivity
> 
> Western blotting is superior to run-of-the-mill IHC in detection 
> sensitivity. This is even more obvious when one considers how easy it 
> is to interpret weak or sparse IHC staining as background. Background 
> is much easier to interpret in a western blot. Therefore an antibody 
> which works beautifully in WB may work in IHC but the proteins might 
> be too sparse or too few in number to be visualized.
> 
> (4) Polyclonal vs. monoclonal
> 
> If an antibody is a polyclonal antibody (pAb) it will likely have a 
> better chance to work across a broad spectrum of applications. This 
> is because a polyclonal antibody preparation is actually a 
> combination of different Ig's recognizing more than one epitope 
> (although it is important to know that there may be a predominant Ig 
> present or the pAb could have been purified in a way to enrich for a 
> particular antigen which may alter it's ability to work in a broad 
> range of apps).
> 
> That's all I can think of for now, and I hope this makes sense ... 
> but as someone else said, it's all empirical and has to be tested in 
> each person's lab.
> 
> Happy New Year!
> 
> 
> >If anyone replies with posting to the list, please forward it.  I've always
> >wondered the same thing.
> >
> >On Fri, Jan 2, 2009 at 8:15 AM, TF <tifei <@t> foxmail.com> wrote:
> >
> >  > Hi
> >  > Just wondering whether the antibody for WB (only WB, IP on datasheet) can
> >>  be used for IHC use?
> >>  If not, why?
> >>
> >>  Can I just increase the concentration for IHC application?
> >>
> >>  I want to learn more about the underlying production processes.
> >>  Thanks!
> >>
> >>
> >>  2009-01-02
> >>
> >>
> >>
> >  > TF
> 
> -- 
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet

_________________________________________________________________
It’s the same Hotmail®. If by “same” you mean up to 70% faster.
http://windowslive.com/online/hotmail?ocid=TXT_TAGLM_WL_hotmail_acq_broad1_122008