[Histonet] Differences between WB antibody and IHC antibody

[Andrea T. Hooper]

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

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