In article <377BA015.37D7B5E1 at sph.unc.edu>, John Scott Meschke <jmeschke at sph.unc.edu> wrote:
:All-
: We are in the process of setting up production of CY-3 labled IgG2
:antibodies. We are rather unexperienced in this technique and need any
:help we can get. Our specific questions revolve around: 1) how purified
:the monoclonals must be prior to lableling,
This depends on your intended use of the monoclonals... Generally, as
pure as possible. My test is as follows: load 5 ug of Ab/lane, stain
with Coomasie - if you don't see _any_ other bands on the gel besides
heavy and light chains, it is "very pure".
:2) what methods of
:purification are best,
You don't specify the source (ascites or growth medium; with or without
serum?) or and the scale of preparation (1 mg or 1 g?) or potential cost
limitations (Affinity step on Protein G is great but rather expensive).
Thus, no answer is really possible.
3) what are the best kits or generic methods for
:labling (at this point, we intend on trying research organics kit),
Have no idea about CY-3. I would assume they are all very similar.
:4)
:what advice do you have for setting up the cell lines, harvesting
:monoclonals, etc. We hope to find a safe, rather idiot-proof set of
:protocols. Thank you in advance for any help or references you may
:give. Thanks again.
Check out some books on antibodies and some companies' booklets
on various options. My default route for small scale
purigication:
- 40% ammonium sulphate precipitation to get rid of fat and
a lot of smaller proteins.
- Ion exchange chromatography on Mono S or Sepharose-SP or something
similar (generally in MES pH around 6.0-6.5, gradient of 10-300 mM NaCl;
can use step elution procedure and gravity flow columns - when optimized
in preliminary experiments).
- Polishing step on Antigen affinity column or - when only one type of
antobodies is present - Protein A (or G if subclass does not bind A well;
don't remember about IgG2).
I prefer ascites but serum-free medium (if you can make cells happy in
it) is also an option, and the only one for large-scale applications.
- Dima
