In article <329506D2.737F at inav.net>, rjjensen at inav.net wrote:
> I am wanting to make fab fragments from a rat IgG2a that was purified
> from a serum free hybridoma supe. Pierce Chemical sells immobilized
> pappain, but I am afraid the fab fragment after digestion will be
> difficult to purify via Protein G. If any of you could send me a
> reference for making fabs from rat IgG2a I would greatly appreciate your
> help. The only reference I have is from 1983 (before the advent of
> Protein G) My E-mail address is rjjensen at inav.net Thanks for your help.
I would very much suggest you to get the following reference:
Goding (1986). Monoclonal Antibodies: principles and practice.
(Academic Press: San Diego, CA, USA).
This book served me very much in preparing Fab fragments from a mouse
IgG1 mAb. Curiously, the Fab fragments still bound to ProteinG, together
with the undigested Ig, and I had to repurify it by ProteinA yielding
the expected separation into Fab's (with did not bind) and Ig (that bound).
In any case, there is really no need to buy a kit for this. Rather you can
work with some cheap papain (might be standing in someones fridge around the
corner) and this wont change too much (just some inactivation step to avoid
the papain to digest your ProteinA/G column that takes 30' at RT by adding
iodoacetamid).
Here is a short protocol:
Papain digestion of T3.70 (mouse IgG1) mAb
The T3.70 mAb was prepared using ProteinA or ProteinG. The digestion
was first performed with a small amount and for different times (Fig. 8).
The optimal duration of the digest was determined using SDS-PAGE
(sodiumdodecyl-sulfate-polyacrylamid-gel-electrophoresis) and staining of
the B6.2.16 T cell clone. The binding of the digested mAb was revealed by
Santi-mIgFITC and measured on the FACScan. SDS-PAGE was performed
initially at non- and reducing conditions. At the non-reducing conditions
the Ab seemed to get partially reduced by cystein in the reaction mixture
needed to hold the papain reduced and active. The information obtained was
therefore difficult to interpret. A large digestion was made using the
same papain solution stored max. 3 days at +4°C. The digest was dialysed
thereafter as needed to purify the Fab fragments from the undigested mAb
by affinity chromatography using ProteinA (Fig. 7) or ProteinG (Fig. 8)
followed by ProteinA. The fragments may then be labeled with a
fluorochrome. Digestion was as described in Harlow and Lane 1988. All
buffers were degassed and the reaction was in a sealed tube under N2 to
prevent oxidation of the papain.
about 5mg/ml Ab (normally the Ab was at a concentration 1-2mg/ml after
ProteinG and was used as such) in 0.1M Na acetate pH 5.5
add 1/20 volume of 1M cysteine 15mg cysteine-hydrochloride
(50mM final conc.) add 100ml 0.1M Na acetate pH 5.5
add 1/20 volume of 20mM EDTA 4ml 0.5M EDTA pH 8.0
(1mM final conc.) ad 100ml 0.1 M Na acetate pH 5.5
add 10mg papain/mg Ab in 0.1 M Na acetate pH 5.5 (1mg papain/ml was practical)
seal tube under N2, mix, incubate at 37°C, for T3.70 1-8h were tryed first
add 1/10 volume of 750mM iodoacetamide 13.875mg iodoacetamide
add 100ml 0.1 M Na acetate pH 5.5
incubate 30' at RT to allow iodoacetamide to react with the papain
The latter step is important since otherwise the papain digests the
affinity column. Digest was stored at +4°C until SDS-PAGE, staining of
B6.2.16 or purification .
To protect the papain from metals EDTA may have been used at a higher
concentration (e.g. 3-4mM). In some other protocols 1mM DTT
(dithiotreitol) is used instead of cysteine to activate the papain before
the digest (Goding 1986).
Good luck,
jorg
---------------------------------------------------------------------
Joerg Kirberg Tel. 0031 - 20 - 512 19 98
The Netherlands Cancer Institute FAX 0031 - 20 - 512 20 11
Div. Molecular Genetics (H4) E_mail kirberg at nki.nl
Plesmanlaan 121
NL - 1066 CX Amsterdam
The Netherlands
