Introduction

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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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). 

Please see our website for how to determine the DOL and additional tips on labeling and reactive reagent choices

Ordering Information

Catalog # Name Size List Price (USD) Qty
A20000 Alexa Fluor® 488 NHS Ester (Succinimidyl Ester) 1 mg 269.00
A20106 Alexa Fluor® 647 NHS Ester (Succinimidyl Ester) 5 mg 1,058.00
A20006 Alexa Fluor® 647 NHS Ester (Succinimidyl Ester) 1 mg 269.00
A20100 Alexa Fluor® 488 NHS Ester (Succinimidyl Ester) 5 mg 1,058.00
T6134 Texas Red®-X, Succinimidyl Ester, mixed isomers 5 mg 221.00
A30005 Alexa Fluor® 488 5-TFP (Alexa Fluor® 488 Carboxylic Acid, 2,3,5,6-Tetrafluorophenyl Ester), 5-isomer 1 mg 269.00
A20009 Alexa Fluor® 555 NHS Ester (Succinimidyl Ester) 1 mg 273.00
A20004 Alexa Fluor® 594 NHS Ester (Succinimidyl Ester) 1 mg 269.00
A20011 Alexa Fluor® 750 NHS Ester (Succinimidyl Ester) 1 mg 269.00
A20002 Alexa Fluor® 546 NHS Ester (Succinimidyl Ester) 1 mg 269.00
A20003 Alexa Fluor® 568 NHS Ester (Succinimidyl Ester) 1 mg 273.00
C6126 6-ROX, SE (6-Carboxy-X-Rhodamine, Succinimidyl Ester), single isomer 5 mg 289.00
C1171 TAMRA, SE; 5-(and-6)-Carboxytetramethylrhodamine, Succinimidyl Ester (5(6)-TAMRA, SE), mixed isomers 25 mg 243.00
A30052 Alexa Fluor® 488 5-SDP Ester (Alexa Fluor® 488 Sulfodichlorophenol Ester) 1 mg 275.00
A20108 Alexa Fluor® 680 NHS Ester (Succinimidyl Ester) 5 mg 1,058.00
A20005 Alexa Fluor® 633 NHS Ester (Succinimidyl Ester) 1 mg 269.00
A20008 Alexa Fluor® 680 NHS Ester (Succinimidyl Ester) 1 mg 269.00
C2211 5-TAMRA, SE (5-Carboxytetramethylrhodamine, Succinimidyl Ester), single isomer 5 mg 283.00
A20103 Alexa Fluor® 568 NHS Ester (Succinimidyl Ester) 5 mg 1,058.00
A20102 Alexa Fluor® 546 NHS Ester (Succinimidyl Ester) 5 mg 1,058.00
T20175 Texas Red®-X, Succinimidyl Ester, single isomer 2 mg 215.00
C6125 5-ROX, SE (5-Carboxy-X-Rhodamine, Succinimidyl Ester), single isomer 5 mg 288.00
C1311 5(6)-FAM, SE (5-(and-6)-Carboxyfluorescein, Succinimidyl Ester), mixed isomers 100 mg 166.00
A20001 Alexa Fluor® 532 NHS Ester (Succinimidyl Ester) 1 mg 269.00
A20010 Alexa Fluor® 700 NHS Ester (Succinimidyl Ester) 1 mg 269.00
A10168 Alexa Fluor® 350 NHS Ester (Succinimidyl Ester) 5 mg 214.00
A20110 Alexa Fluor® 700 NHS Ester (Succinimidyl Ester) 5 mg 1,058.00
A20109 Alexa Fluor® 555 NHS Ester (Succinimidyl Ester) 5 mg 1,058.00
A20104 Alexa Fluor® 594 NHS Ester (Succinimidyl Ester) 5 mg 1,058.00
A10169 Alexa Fluor® 430 NHS Ester (Succinimidyl Ester) 5 mg 214.00
C6171MP 6-JOE, SE (6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein, Succinimidyl Ester) 5 mg 279.00
Q10193 QSY® 7 Carboxylic Acid, Succinimidyl Ester 5 mg 225.00
A20111 Alexa Fluor® 750 NHS Ester (Succinimidyl Ester) 5 mg 1,058.00
T2556 Tris-(2-Carboxyethyl)phosphine, Hydrochloride (TCEP) 1 g 89.00
Q20132 QSY® 21 Carboxylic Acid, Succinimidyl Ester 5 mg 229.00
P30253 Pacific Orange™ Succinimidyl Ester, Triethylammonium Salt 1 mg 231.00
A20101MP Alexa Fluor® 532 NHS Ester (Succinimidyl Ester) 5 mg 1,058.00
F6130 Fluorescein-5-EX, Succinimidyl Ester 10 mg 138.00
C6123 6-TAMRA, SE (6-Carboxytetramethylrhodamine, Succinimidyl Ester), single isomer 5 mg 294.00
A30051 Alexa Fluor® 790 NHS Ester (Succinimidyl Ester) 100 µg 168.00
C2210 5-FAM, SE (5-Carboxyfluorescein, Succinimidyl Ester), single isomer 10 mg 211.00
C6164 6-FAM, SE (6-Carboxyfluorescein, Succinimidyl Ester), single isomer 10 mg 209.00
A30100 Alexa Fluor® 405 NHS Ester (Succinimidyl Ester) 5 mg 1,058.00
A20105 Alexa Fluor® 633 NHS Ester (Succinimidyl Ester) 5 mg 1,058.00
R6160 Rhodamine Red™-X, Succinimidyl Ester, 5-isomer 5 mg 168.00
C20050 CMNB-Caged Carboxyfluorescein, SE (5-Carboxyfluorescein-Bis-(5-Carboxymethoxy-2-Nitrobenzyl) Ether, β-Alanine-Carboxamide, Succinimidyl Ester) 1 mg 169.00
Q20131 QSY® 9 Carboxylic Acid, Succinimidyl Ester 5 mg 229.00
C6157 5(6)-CR 6G, SE (5-(and-6)-Carboxyrhodamine 6G, Succinimidyl Ester), mixed isomers 5 mg 172.00
A30002 Alexa Fluor® 514 NHS Ester (Succinimidyl Ester) 1 mg 269.00
C6156 6-ROX (6-Carboxy-X-Rhodamine), single isomer 10 mg 208.00
S1534 SMCC, Succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-Carboxylate 100 mg 209.00
S1531 Succinimidyl 3-(2-Pyridyldithio)Propionate (SPDP) 100 mg 232.00
F6106 5-SFX (6-(Fluorescein-5-Carboxamido) Hexanoic Acid, Succinimidyl Ester), single isomer 5 mg 138.00
C6124 5-ROX (5-Carboxy-X-Rhodamine, Triethylammonium Salt), single isomer 10 mg 210.00
C300 5(6)-TAMRA (5-(and-6)-Carboxytetramethylrhodamine), mixed isomers 100 mg 172.00
C1309 5(6)-ROX, SE (5-(and-6)-Carboxy-X-Rhodamine, Succinimidyl Ester), mixed isomers 25 mg 248.00
S1167 NBD-X, SE, Succinimidyl 6-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino)Hexanoate 25 mg 209.00
C1359 5-FAM (5-Carboxyfluorescein), single isomer 100 mg 138.00
C1360 6-FAM (6-Carboxyfluorescein), single isomer 100 mg 137.00
M10165 Marina Blue® Succinimidyl Ester 5 mg 228.00
A30050 Alexa Fluor® 610-X NHS Ester (Succinimidyl Ester) 1 mg 269.00
C6127 5-CR 6G, SE (5-Carboxyrhodamine 6G, Succinimidyl Ester), single isomer 5 mg 259.00
B1513 D-Biotin, Succinimidyl Ester (succinimidyl D-Biotin) 100 mg 104.00
H2249 NHSS (N-Hydroxysulfosuccinimide, Sodium Salt) 100 mg 66.00
D10161 Dapoxyl® Carboxylic Acid, Succinimidyl Ester 5 mg 128.00
C653 5-(and-6)-Carboxynaphthofluorescein, Succinimidyl Ester, mixed isomers 25 mg 114.00
S6110 PyMPO, SE, 1-(3-(Succinimidyloxycarbonyl)Benzyl)-4-(5-(4-Methoxyphenyl)Oxazol-2-yl)Pyridinium Bromide 5 mg 209.00
C6121 5-TAMRA (5-Carboxytetramethylrhodamine), single isomer 10 mg 208.00
C6122 6-TAMRA (6-Carboxytetramethylrhodamine), single isomer 10 mg 208.00
LT116          15-Nov-2010