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IP Problem

IP Problem - Protocols and Methods Forum

IP Problem - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 07-10-2008, 05:43 PM
John Leonard
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Default IP Problem



Hello all,

I'm looking to perform an IP Western of a protein that runs at around
50-55kD on an SDS-PAGE gel - approximately the same as my antibody's heavy
chain. As such, it could be very difficult to detect presence of my protein
by IP Western, since the antibody is generally eluted along with the
antigen. I have read protocols which suggest to cross-link your antibody to
the protein A/G beads prior to IP, however I'm using rabbit antiserum (not
immunoaffinity purified), so I don't know whether this will be effective.
A co-worker of mine suggested running a non-denaturing gel instead to
observe either a) separation of the protein of interest and the antibody by
nature of different charge/size ratios; or b) differential shifting of
the antibody band (when compared with a negative/positive controls) to
indicate interaction with my protein of interest. I don't know how
effective this would be, but in that case I would need a good NON-DENATURING
elution buffer to detach my antigen from the bead complex.

The whole purpose of this experiment is to determine whether my antiserum
will be sufficient for IPs, ChIP, etc or whether I should immunoaffinity
purify it. (I'll be pre-clearing with normal rabbit serum to reduce
non-specific binding).

If anyone has comments/suggestions about this, or knows of A GOOD
NON-DENATURING ELUTION BUFFER, I'd love to hear it!


Thanks,

John
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  #2  
Old 07-10-2008, 08:33 PM
allisonh
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Default IP Problem

John Leonard wrote:

If you run a regular SDS gel without reducing agent in the sample buffer
(ie, no beta-mercaptoethanol) then the antibodies will run at 150K. If
your protein is a single unit then it will still run at about 50-55K.


Allison
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  #3  
Old 07-10-2008, 09:07 PM
DK
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Default IP Problem

In article <[Only registered users see links. ].net >, "John Leonard" <[Only registered users see links. ]> wrote:

You mix protein A/G sepharose with serum, rigorously wash, then
cross-link. It's being done routinely. In my limited experience,
though, the resulting immunoadsorbent never works as efficiently
as "homogenous" IP (react IgG with antigen, then pull everything down
with immobilized protein A/G).


If you want clean results you can trust and hope to convince others
to trust, you simply must use IgG purified against your antigen.
There are too many IP results of dubuous quality and value already
published - no need to increase optical density of the literature.

DK
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  #4  
Old 07-10-2008, 10:42 PM
AK
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Default IP Problem

I had same problem once added with my co-IP protein died upon heating the
sample. 6M urea in sample buffer did the trick without heating which sent
the IgG bands close to 100kDa. if I remember correctly I also added 10mM DTT
that apparently did not effect shift of IgG to higher mol wt signal. good
luck.
AK
"John Leonard" <[Only registered users see links. ]> wrote in message
news:[Only registered users see links. ].n et...


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  #5  
Old 07-11-2008, 06:36 PM
Dr Engelbert Buxbaum
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Default IP Problem

Am 10.07.2008, 13:43 Uhr, schrieb John Leonard <[Only registered users see links. ]>:


This largely deppends on how you want to detect your protein after
electrophoresis. Usually in IP you have too small an amount to detect by
protein staining. Instead the target protein is usually metabolically
labelled (originates from cell cultures grown with 35-S Met/Cys), the
presence of Ig heavy chain would not interfere with detection as it is
non-radioactive.

A little technical hint: Protein A beads are quite expensive. For IP I use
fixed Staph. aureus cells instead. These are commercially availble (e.g.
Pansorbin from Calbiochem).
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  #6  
Old 07-11-2008, 09:41 PM
Jayakumar, R
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Default IP Problem

Not very difficult at all. Use a different antibody (for e.g. Mouse)
for IPing the protein down and use another one for western detection
(for e.g. Rabbit). Then the heavy chain of mouse will not be visible
when we use anti-Rabbit secondary for western detection.

Jay

-----Original Message-----
From: [Only registered users see links. ]
[mailto:[Only registered users see links. ].indiana.edu] On Behalf Of Dr Engelbert
Buxbaum
Sent: Friday, July 11, 2008 2:37 PM
To: [Only registered users see links. ]
Subject: Re: IP Problem

Am 10.07.2008, 13:43 Uhr, schrieb John Leonard <[Only registered users see links. ]>:



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