Production of IgM monomers

Mike Clark mrc7 at cam.ac.uk
Fri May 31 07:32:19 EST 2002


In article <0DE50F4E1F75D411803C00508BE70B2D57BE07 at KSB-GUILDFORD>,
Richard Truran <URL:mailto:RichardTruran at KSBiomedix.com> wrote:
> I am researching the activity of a mouse IgM monoclonal and a mouse IgG,
> they both recognise the same antigen, I want to investigate the effect of
> valency, and so want to be able to break the IgM molecule into the
> monomeric form. Is this possible and if so does anyone know how.
> 
> Thanks
> 
> Richard Truran 
> 

You need to look at the papers of Arnold Fienstein from the 70s (He did a
lot of the pioneering work on the structure of IgM). I used a method of his
for my PhD work in 78-81.

Buffer 0.3M NaCl 0.2M TrisHCl pH 8.0 10mM EDTA

(pH8.0 pKa of cysteine)  Reduce with 10mM DTT (equiv of 20mM thiol) for 1
hour at room temeperature.

Alkylate with  iodoacetamide at a 50% M excess over thiol for 1 hour at
room temperature in the dark.

Dialyse against PBS.



Mike                           <URL:http://www.path.cam.ac.uk/~mrc7/>
-- 
M.R. Clark, PhD. Division of Immunology
Cambridge University, Dept. Pathology
Tennis Court Rd., Cambridge CB2 1QP
Tel.+44 1223 333705  Fax.+44 1223 333875




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