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antibody purification - (Nov/01/2005 )

Could anyone give any advice how to purify antibodies. I have polyclonal antibodies against peptides and full-length proteins and I have already purified them with proteinG columns. But they still don't work in cell samples (lot of background in Western but not the correct bands), only against recombinant proteins. I have also tried to purify them further with CNBr-activated Sepharose 4B, but the antibodies bind to sepharose really tight and I can't elute them out sad.gif

-Kati

-Kati-

QUOTE (Kati @ Nov 1 2005, 02:32 PM)
Could anyone give any advice how to purify antibodies. I have polyclonal antibodies against peptides and full-length proteins and I have already purified them with proteinG columns. But they still don't work in cell samples (lot of background in Western but not the correct bands), only against recombinant proteins. I have also tried to purify them further with CNBr-activated Sepharose 4B, but the antibodies bind to sepharose really tight and I can't elute them out sad.gif

-Kati


You hit a tough problem Kati ! If your antibodies are very affine they will bind like crazy and therefore you will need harsh conditions to get them out of your column. Some Ab's doesn't like too much acidic treatment

One or two questions

1/ When you purify them through Protein G which buffer did you use to elutae them ?

2/ At wich pH ?

3/ Did you neutralise the fractions right away ?

pesji cool.gif

-pesji-

hi,

if you're still getting high background after prot G purification, try some different affinity purification strategies. here are two, both variations on immunoaffinity adsorption:

1.) purify some recombinant protein (i'm guessing you have an E. coli expression system to make your antigen). pour a preparative SDS-PAGE gel and run out a mg or two. blot this to nitrocellulose (for some reason, PVDF does not work so well w/ this protocol). stain w/ ponceau and cut out your band w/ a scalpel. block w/ 5% carnation in 1X PBS-T for 1hr at room temp. wash this w/ 1X PBS to remove excess blocker and then take 1ml of your crude serum, dilute it in 5ml 1X PBS, and let the strip and Ab solution incubate in a 15ml conical tube at room temp for 2 hours. remove the Ab solution, wash 3X, at 5 min each w/ 1X PBS-T and elute 5ml w/ 100mM glycine, pH 2.0 at room temp for 5 min. pour your buffer into a new tube and add 500┬Ál 1M tris, pH 8.0 to neutralize it. after that, run several dilutions against your crude extract and possibly 1-5ng of your recombinant protein and see if your signal:noise ratio improves.

2.) couple your purified recombinant protein to Affigel10 or affigel 15 (depending on it's pI). this method is also quick and easy, though it requires you to dialyze your protein into an appropriate coupling buffer after purification. once bound to the resin, incubate your protein w/ it as above in a 15ml tube, or in a Bio Rad polyprep column (be sure not to open the bottom cap!) on an end-over shaker at room temp for 2hr. remove the bottom cap and elute w/ glycine as above, and don't forget to neutralize as above.

coupling your peptide to CNBr resin is a bit of a hassle, and often small peptides are chemically unstable. this makes using purified recombinant protein a more desirable antigen in purifying Abs from crude serum. try either of the above methods, and see if things don't improve. if you have a choice, go w/ method #2, as you'll get better yield and somewhat cleaner Abs, but method 1 will work fine if you're just looking to see proteins on an immunoblot.

if you have further questions, feel free to email me.

good luck,

jon

jonmike.reed@gmail.com

QUOTE (pesji @ Nov 1 2005, 08:46 AM)
QUOTE (Kati @ Nov 1 2005, 02:32 PM)

Could anyone give any advice how to purify antibodies. I have polyclonal antibodies against peptides and full-length proteins and I have already purified them with proteinG columns. But they still don't work in cell samples (lot of background in Western but not the correct bands), only against recombinant proteins. I have also tried to purify them further with CNBr-activated Sepharose 4B, but the antibodies bind to sepharose really tight and I can't elute them out sad.gif

-Kati


You hit a tough problem Kati ! If your antibodies are very affine they will bind like crazy and therefore you will need harsh conditions to get them out of your column. Some Ab's doesn't like too much acidic treatment

One or two questions

1/ When you purify them through Protein G which buffer did you use to elutae them ?

2/ At wich pH ?

3/ Did you neutralise the fractions right away ?

pesji cool.gif

-johanski-

I used 0.1 M glycine-HCl, pH 2.7 as an elution buffer and yes, I did neutralize them right away.

-Kati

QUOTE (pesji @ Nov 1 2005, 03:46 PM)
QUOTE (Kati @ Nov 1 2005, 02:32 PM)

Could anyone give any advice how to purify antibodies. I have polyclonal antibodies against peptides and full-length proteins and I have already purified them with proteinG columns. But they still don't work in cell samples (lot of background in Western but not the correct bands), only against recombinant proteins. I have also tried to purify them further with CNBr-activated Sepharose 4B, but the antibodies bind to sepharose really tight and I can't elute them out sad.gif

-Kati


You hit a tough problem Kati ! If your antibodies are very affine they will bind like crazy and therefore you will need harsh conditions to get them out of your column. Some Ab's doesn't like too much acidic treatment

One or two questions

1/ When you purify them through Protein G which buffer did you use to elutae them ?

2/ At wich pH ?

3/ Did you neutralise the fractions right away ?

pesji cool.gif

-Kati-

[quote name='Kati' date='Nov 10 2005, 03:49 PM' post='30639']
I used 0.1 M glycine-HCl, pH 2.7 as an elution buffer and yes, I did neutralize them right away.

-Kati




Hi
do you have some protein precipitation when you neutralize the glycine pH 2.7?

-velumax-

[quote name='velumax' date='Nov 17 2005, 02:36 PM' post='31554']
[quote name='Kati' date='Nov 10 2005, 03:49 PM' post='30639']
I used 0.1 M glycine-HCl, pH 2.7 as an elution buffer and yes, I did neutralize them right away.

-Kati




Hi
do you have some protein precipitation when you neutralize the glycine pH 2.7?
[/quote]


At least I haven't notice any.

-Kati

-Kati-