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Protein purification using mouse anti-Myc Tag IgG

Protein purification using mouse anti-Myc Tag IgG - Protocols and Methods Forum

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  #1  
Old 12-21-2006, 05:18 PM
Breslauer
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Default Protein purification using mouse anti-Myc Tag IgG



Protein purification using mouse anti-Myc Tag IgG
Hi there,

have question about publications & your experience. I'm going to purify
partially purifed recombinant protein (but containing ~30% of protein
contaminations) using affinity chromatography. I produced a lot of
monoclonal antibody - mouse anti-myc, and my protein has Myc-Tag.
Saying produced I mean collected supernatant from hybrydoma cells. The
thing is to purify & bind my antibodies and (it would be great if using
the same resign/column) pass my protein throuh it, hoping it will bind
with antibodies. Then wash & elute it. Do u thing that sepharose with
protein A will be good? Does any of u who have expereince in this
experiment may discuss this idea of expriment with me? Waiting for your
replay.

Bres

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  #2  
Old 12-21-2006, 06:00 PM
Nick Theodorakis
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Default Protein purification using mouse anti-Myc Tag IgG


Breslauer wrote:

Is your culture supernatant from serum-free media, or does it contain a
lot of bovine anitbodies?

Nick

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  #3  
Old 12-22-2006, 06:09 AM
Breslauer
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Default Protein purification using mouse anti-Myc Tag IgG

Dear Nick,

I use D-MEM or RPMII serum free media.
Bres

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  #4  
Old 12-22-2006, 11:05 PM
Pow Joshi
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Default Protein purification using mouse anti-Myc Tag IgG

On 21 Dec 2006 22:09:00 -0800, Breslauer <[Only registered users see links. ]> wrote:

ah, if you use serum free medium, then Protein A sepharose whould work
very well for mouse IgG.

best,
pow

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  #5  
Old 12-23-2006, 06:51 PM
Pow Joshi
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Default Protein purification using mouse anti-Myc Tag IgG

Hi Eberhard,

I found the table from Sigma ...and yes, it does say that for IgG1 the
binding is better with Protein G and L; and I presume you have the
IgG1 isotype.
I will give you the procedure I had used, a long time ago for such
purifications: However, for more details you could refer to Current
protocols in immunology and/ or Antibodies by Ed Harlow and David
Lane.

The way I went about my purifications was:
1. 45% ammonium sulphate cut off (use the precipitate for further
purifications, since that is where the bulk of the antibodies are)
2. I had added a step of DEAE sepharose step (I was using serum)
....however, I suppose you can delete this step.
3. I had made a small column (of about 1cm diameter and 4 cm height,
which would roughly make about 4 ml of bed volume) and equiliberated
the column with 10mM phosphate buffer, pH 7; applied the antibody
solution from step 2 on teh column; alllowed the antibody fraction to
bind for about 1.2 hr. Eluted the antibody with 100mM glycine-HCl
buffer, pH 2.7 (you will have to neutralise the eluate, otherwise the
antibody will be inactivated!)
You can concentrate this fraction further with ultrafiltration.
You will have >=95% purity.

I would imagine, that for you, even the ammounium sulphate
precipitation should work well, since you have serum free culture
supernate, and the only protein there would be the monoclonal released
(perhaps a few cytokines maybe?)
let me know if you'd have any further questions.....and hope that works for you.

pow


On 12/23/06, Breslauer <[Only registered users see links. ]> wrote:

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