MSD GOLD™ SULFO-TAG NHS-Ester
MSD GOLD SULFO-TAG™ NHS-Ester  150 nmol (sufficient for conjugating 1 mg of IgG)  R91AO-1
2 µmol (sufficient for conjugating 10 mg of IgG)  R91AO-2
MSD GOLD SULFO-TAG NHS-Ester Conjugation Packs  Pack 1 (sufficient for conjugating 5 x 200 µg of IgG)  R31AA-1 Pack 2 (sufficient for conjugating 5 x 1 mg of IgG)  R31AA-2
MSD Labeling Reagents
MSD GOLD SULFO-TAG NHS-Ester
For labeling amines
Note:
150 nmol size of MSD GOLD SULFO-TAG NHS-Ester is sufficient for conjugating 1 mg of IgG and the 2 µmol size is sufficient for conjugating 10 mg of IgG at a challenge ratio of 20.
Each MSD GOLD SULFO-TAG NHS-Ester Conjugation Pack contains enough material for 5 reactions. A
t a challenge ratio of 20, Pack 1 is sufficient for conjugating 200 µg of IgG per reaction, and Pack 2 is sufficient for conjugating 1 mg of IgG per reaction.
This package insert must be read in its entirety before using this product.
FOR RESEARCH USE ONLY.
NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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Table of Contents
Introduction (4)
Preparation of MSD GOLD SULFO-TAG Conjugates (5)
MSD GOLD SULFO-TAG NHS-Ester Conjugation Packs (6)
Additional Materials and Equipment (6)
Protocol (8)
Storage, Handling, and Stability (10)
reactive toFAQs (11)
Worksheet (14)
Ordering Information
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Email: ScientificSupport@mesoscale
Introduction
This protocol details the conjugation procedure for proteins of molecular weight (MW) > 40,000 D altons using MSD GOLD SULFO-TAG NHS-Ester label. The straightforward procedure involves an optional buffer exchange step, a 2-hour incubation step, and a mandatory buffer exchange step to quickly isolate the conjugated protein using a spin column. MSD GOLD SULFO-TAG NHS-Ester (Figure 1) is an amine reactive, N-hydroxysuccinimide ester which readily couples to primary amine groups of proteins under mildly basic conditions to form a stable amide bond.
MSD GOLD SULFO-TAG conjugates are stable and may be used at low concentrations. These features minimize time, cost, and labor as large batches of a stable conjugate can be prepared, validated, and used for long periods of time. Its excellent performance characteristics and simple conjugation procedure make MSD GOLD SULFO-TAG NHS-Ester the product of choice for molecules that contain primary amines (e.g., lysine-containing proteins). MSD GOLD SULFO-TAG offers low non-specific binding, resulting in highly sensitive detection when used in conjunction with MSD instruments.
Figure 1: MSD GOLD SULFO-TAG NHS-Ester
Preparation of MSD GOLD SULFO-TAG Conjugates
General Notes
In order to minimize hydrolysis of MSD GOLD SULFO-TAG NHS-Ester, the reagent should be dissolved in cold distilled water just prior to its addition to the protein solution. If necessary, the stock MSD GOLD SULFO-TAG NHS-Ester solution can be kept on ice for up to 10 minutes. The reconstituted solution is unstable and any unused material should be discarded. Consider conjugating more than one protein at the same time to maximize the use of the MSD GOLD SULFO-TAG NHS-Ester reagent. Generally, 150 nmol of MSD GOLD SULFO-TAG NHS-Ester is sufficient for conjugation of up to 1 mg of IgG.
The labeling ratio is the number of molecules of MSD GOLD SULFO-TAG conjugated to each molecule of protein. Optimal conjugation ratios for a MSD GOLD SULFO-TAG conjugated protein should be determined empirically for each specific application. For most applications using IgG antibodies (MW ~150,000), optimal performance is obtained with conjugation ratios between 2:1 and 20:1. In this range, assay signals typically show a linear dependence on conjugation ratio. Conjugation ratios significantly higher than 10:1 can be counterproductive and may lead to elevated background sig
nals or loss of binding activity. For proteins that are significantly smaller than IgGs, lower conjugation ratios (between 1:1 and 5:1) may provide better assay performance.
The challenge ratio is the number of moles of MSD GOLD SULFO-TAG per mole of protein in the conjugation reaction mixture. The challenge ratio required to achieve a specific conjugation ratio depends on a number of factors including pH, temperature, protein concentration, protein size, and the number of lysines available for coupling. Conjugating a 2 mg/mL IgG solution using the standard conditions described in this protocol will typically result in a label incorporation of approximately 50% (i.e., a challenge ratio of 10:1 will result in a conjugation ratio of about 5:1). Conjugation efficiencies for other proteins may be different. In general, conjugating with high protein concentrations of 1–2 mg/mL in slightly alkaline PBS (pH 7.9) in the absence of preservatives yields the best conjugation efficiencies. Maintaining consistent conjugation conditions (protein concentration, buffer type, MSD GOLD SULFO-TAG NHS-Ester concentration, incubation time, and temperature) is important when preparing multiple batches of conjugated protein in order to achieve consistent assay results.
When developing immunoassays, MSD recommends conjugating antibodies using the standard conditions outlined in this document and challenge ratios of 6:1, 12:1, and 20:1 to identify optimal conjugation conditions. If evaluating different conditions is not possible due to limited reagent quantitie
s, a challenge ratio of 20:1 will generally provide good performance. For immunogenicity applications where an antibody drug or protein therapeutic is used, the suggested challenge ratios are 12:1 and 6:1 MSD GOLD SULFO-TAG:drug. If only one ratio is tested, a 10:1 challenge ratio is recommended. For details on building immunogenicity assays, please refer to the Bridging Immunogenicity Guidelines for Assay Development soscale. The protocol describes the MSD GOLD SULFO-TAG conjugation procedure for proteins with a MW > 40,000 D a. Smaller proteins/polypeptides may also be conjugated using MSD GOLD SULFO-TAG NHS-Ester as long as they have an accessible lysine or the N-terminal amino group; however, alternative separation methods may be needed to remove unconjugated label. MSD offers a variety of services for the custom conjugation of reagents including proteins, peptides, and non-proteinaceous molecules.