Polyclonal antibody purification? - how? (Dec/01/2006 )
I just got anti serum, which gives a lot of background bands on my blot (some of them are even more intense than the antigen one.)... Can I purify it with some "easy" methods?
Thank you .
you could use protein A or protein G sepharose column.
here is a protocol, just for example.
Protein A or G are quite easy but may be rather expensive for some labs. (Like mine... )
Ammonium sulphate precipitation is quite good, easy and cheap (you may also try caprylic acid first but some say it won't improve quality that much).
Missele, I thought about the proteinA sepharose, but will it pull down other Igs in the serum?
K.B. It also might be expensive for my lab, I have to ask. But what is the Ammonium sulphate precipitation? Sorry that I have no knowledge about it. I will look for the protocol.
yes, it will pull down other Ig, but sulphate ammonium also.
If you really want to purify the specific antigen, you need to prepare a column with the antigen ( if I remember well you need something like 30 mg of purifed antigen), and CnBr activated sepharose to couple the antigen to the column;
tell me that you already tried to dilute more your primary and secondary antibodies.
what's your protocol? I mean, do you incubate in TBS, TBS-tween? do you add BSA or milk during incubation with antibody?
you could also increase the concentration of NaCl up to 500 mM in the washing buffer and/or in the incubation buffer
We don't have purified antigen yet...
The dilution of serum was 1:2000 (Rabbit serum in 1% milk in PBS-Tween), 2nd I used GARPO, 1:50,000.
I used a very simple lysis buffer, PBS (from cell culture room) with 0.5% NP40, the blot was blocked in 5% milk (without BSA), antibodies were diluted in 1% milk as I typed, all the washing steps except the last one or two were using 1% milk in PBS-T.
I will try blocking buffer with 5%milk and 0.5%BSA next week, which is a suggestion of my colleague. If that doesn't work, I do want to purify this antibody..
1 mL of protein A sepharose would buy you about 1 kg of molecular biology grade >99% ammonium sulphate (whis is enough to precipitate more than 1 L of serum).
Protein A or G are much better so if you have time and money do not hesitate.
your protocol sounds good.
But, have ever tried the secondary antibody without the primary antibody on YOUR sample? (Just to be sure that the non specific bands are due to primary.)
you could also try to block in 3% BSA, incubate the primary in 3% BSA (and try also increasing dilutions of the primary) and then incubate the secondary in the presence of milk.
This protocol works fine for most of my antibodies, but unfortunately there is no general rule. You have to try.
I don't know if protein A purification will help, it could be due to Ig?
so your issues are :
- find a good WB protocol for this antibody
- purify your antibody with antigen coupled CNBr column?
Do you still have the antigen you immunized the rabbits with? That is what I conjugate my columns with for affinity purification. Depending on the amount of serum you have to purify, you can get a kit from Pierce that is pretty cheap to conjugate your columns and also they have purification kits too I think.
Thank you so much for all these nice suggestions.
K.B., Thank you for the protocol, I will try if the background is still there after all the combination of blocking buffer.
Missele, I haven't tried the 2nd aloneb because it is thought to be a very good one.. But last time, I used milk with 0.5% BSA, the background seems less. I am going to try other concentrations of BSA.
WAstate: I immunized rabbits with antigen in SDS-PAGE. So, there's no pure antigen protein solution.
Thank you again.