Immunoglobulin M (IgM) antibodies are generally the first to appear in response to initial antigen exposure. IgM is by far the physically largest antibody; it is in essence polymers of individual immunoglobulin molecules covalently linked by disulfide bonds. The common pentameric structure has a molecular mass of approximately 900 kDa.
For researchers interested in labeling IgM antibodies with fluorescent labels, their structural complexity and sensitivity to pH and other physical conditions requires modification of protocols optimized for IgG class antibodies. Here we describe a protocol for IgM labeling that has been optimized in our laboratory with our superior amine-reactive Alexa Fluor® dyes (Alexa Fluor® NHS and SDP esters).
The amine-reactive Alexa Fluor® dyes react with non-protonated aliphatic amine groups (the α-terminus and lysine ε-amino groups). IgG labeling reactions are typically performed at pH 8.3 to 9.0. However, IgM antibodies tend to denature at even slightly alkaline pH values and thus labeling must be done close to neutrality. Labeling is less efficient near neutrality and hydrolysis dominates over protein labeling. At the near neutral pH used to conjugate IgM antibodies, primarily α-amino end-termini will be labeled; lysine ε-amino groups have pKa values around 10.5 and are thus largely protonated and unreactive. For these reasons the dye:IgM molar ratio needs to be on the order of 50:1 or 100:1, rather than the 10:1 ratio generally used for IgG labeling.
The following is a modification of the standard amine reaction protocol that we have developed for labeling of IgG molecules using our amine-reactive dyes and protein labeling kits.
- Prepare your antibody in reaction buffer: 0.1 M sodium phosphate, pH 7.2 to 7.5, with 150 mM NaCl for improved antibody solubility (PBS is ideal). Note: Antibodies supplied in PBS, pH 7.4, with 0.1% (or less) sodium azide can be used directly in the labeling reaction. Buffers containing primary amines (e.g., Tris or glycine) will strongly interfere because they react with the NHS-ester moiety. If your IgM molecule is suspended in such a buffer it must first be dialyzed against reaction buffer.
- Transfer 100-500 μL of antibody (0.5 to 5 mg/mL) to the vial containing the dye. Mix well and incubate at room temperature for 1 hour. As the Alexa Fluor® amine reactive dyes are relatively hydrophilic (especially the SDP ester), it is not necessary to dissolve the dye in organic solvent.
Alternatively, dissolve 1 mg reactive dye in 10 to 100 uL anhydrous DMSO. This corresponds to a concentration of 100 and 10 mg/mL, respectively.
- Remove un-conjugated dye by passing the solution over a short gel filtration column (e.g. BioSpin #732-6008, BioRad). Alternatively dialysis at 2-6 °C for 12 to 24 h with four buffer changes can be used.
- Store the labeled antibody protected from light at 2-6 °C. We generally do not recommend frozen storage of antibody conjugates; if you wish to store your labeled antibody at -20 °C, we suggest adding 50% (v/v) glycerol to single use aliquots.
- For storage at 2-6 °C longer than a few weeks we recommend adding sodium azide (NaN3) to a final concentration of 0.02-0.05% (w/v) (0.3-0.75 mM), which will inhibit microbial growth
In all labeling reactions we recommend testing more than one ratio to achieve optimal degree of labeling (DOL).