Technical information

Antibody types

Purification

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Technical information > IgM

Specific IgM antibodies are a result of a primary response to the challenge with antigen. In some cases, production of polyclonal or monoclonal antibodies for some antigens will result only in one the level of IgM.

Pentameric or hexameric IgM

Molecular mass: ~160 kDa
Extinction coefficient at 280 nm: 1.18 (Johnstone and Thorpe 1988)
Weight of IgM (or other protein) to molar quantity can be converted here

Some important points to consider when working with IgM

IgM is poorly soluble.
IgM has a tendency to cryoprecipitate.
IgM can "stick" to the walls of a tube. This can be prevented by pre-coating with Tween solution or including a carrier protein like FBS or BSA.
Purified IgM might be stored at 4°C, depending upon the antibody, and can be stable for around 6-10 months.
IgM might precipitate in buffers of low ionic strength.
There are possible problems with retaining IgM activity during hydrophobic chromatography.
IgM antibodies often give unexpected cross-reactivities, and it is important to realize that certain problems might occur due to this (Goding 1996)

How to stabilize IgM? Some general considerations

IgM might require higher salt concentration than 150 mM NaCl; try to increase the NaCl concentration up to 350-500 mM NaCl.
Try eluting IgM with a chaotropic salt (3-3.5 M) and exchange into 500 mM NaCl.
Borate buffer is recommended for storage of IgM, its subunits or its fragments (J. E. Coligan et al. 1991, Current Protocols in Immunology).
Glycine has a stabilizing effect on IgM: adding about 0.01 M glycine, should also help. Note: consider a possible influence of a glycine in different assays.
IgM can also be stabilized by addition of BSA or HSA to final 0.05% or 0.5%.
Addition of glycerol might be of a help as well.

Agrisera Secondary antibodies to human IgM

IgM storage conditions

Storage in PBS (including azide in 0.02% final concentration) is recommended. Microbial contamination is very often a cause of lost activity in antibodies. Use a good preservative and make sure that you prepare things aseptically. Freezing of IgM should be tested for each individual antibody. Some IgM antibodies might be inactivated by freezing.

Valuable information about IgM cab be found in:
"Monoclonal antibodies: principles and practice" by James W. Goding, 1996, ISBN 0-12-287023-9; Publisher: Academic Press.

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