Production of IgM monomers
mrc7 at cam.ac.uk
Mon Jun 3 09:19:00 EST 2002
In article <ant3112190b0Pk=+ at mrc7acorn1.path.cam.ac.uk>, Mike Clark
<URL:mailto:mrc7 at cam.ac.uk> wrote:
> 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.
Sorry I got the i and the e the wrong way round!
Here are the papers I meant.
Record 1 of 4 in MEDLINE(R) on CD 1980-1981
TI: Structure of hydrated immunoglobulins and antigen-antibody complexes.
Electron microscopy of spray-freeze-etched specimens.
AU: Munn,-E-A; Bachmann,-L; Feinstein,-A
SO: Biochim-Biophys-Acta. 1980 Sep 23; 625(1): 1-9
AB: The structure of spray-frozen IgG and IgM, either free in solution or
specifically bound to bacterial flagella is compared after freeze-etching
with the corresponding preparations negatively stained. The freeze-etched
IgG is readily detected; most of the hydrated particles appear
approximately spherical with an average diameter of about 12 nm. About 20%
are triangular with sides of about 13-14 nm. Antibodies attached to
flagella are clearly seen on the top as well as the exposed edges. The
technique can thus be used for antigen localization on freeze-etched
macromolecules and also on the surface of larger structures. The dimensions
and shape of freeze-etched bound IgM confirm the earlier interpretations of
the structure of this antibody based on negative staining. AN: 81021728
Record 2 of 4 in MEDLINE(R) on CD 1978-1979
TI: Mouse intracellular immunoglobulin M. Structure and identification of
a free thiol group.
AU: Richardson,-N-E; Feinstein,-A
SO: Biochem-J. 1978 Dec 1; 175(3): 959-67
Record 3 of 4 in MEDLINE(R) on CD 1978-1979
TI: The covalent assembly of MOPC 104 E immunoglobulin M. Identification
of a heavy chain dimer, a complex of two heavy chains and one light chain,
and a distinctive form of monomer as intracellular intermediates.
AU: Percy,-M-E; Feinstein,-A; Baumal,-R
SO: Can-J-Biochem. 1978 Mar; 56(3): 190-6
Record 4 of 4 in MEDLINE(R) on CD 1973-1975
TI: Interchain disulphide bridges of mouse immunoglobulin M.
AU: Milstein,-C-P; Richardson,-N-E; Dieverson,-E-V; Feinstein,-A
SO: Biochem-J. 1975 Dec; 151(3): 615-24
AB: Mouse IgM (immunoglobulin M) was selectively and partially reduced and
treated with iodo[2-14C]acetate to label the interchain disulphide bridges.
The carboxymethylation was studied in some detail. The labelled peptides
were purified, sequenced and positioned by homology with human IgM. Only
peptides originating from three interchain disulphide bridges were
labelled, in contrast with the four labelled bridges obtained in human IgM
under the same conditions. These peptides are homologous to human bridge
peptides forming the heavy-light bridge and two inter-heavy bridges, one
present in the CMU2 region and the other in the C-terminal region. The
inter-heavy bridge in the Cmu2 region was alone cleaved and radioactively
labelled in selectively reduced IgM held together as a pentamer by
non-covalen interactions. The same bridge was the only one to be totally
cleaved in subunits released after more extensive, though still selective,
reduction. In the light of these results a possible arrangement of the
disulphide bridges of the mouse IgM. AN: 76135505
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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