Buccutite™ MTA, NHS ester

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	659.70
Solvent	DMSO

Storage, safety and handling

Intended use	Research Use Only (RUO)
Storage	Freeze (< -15 °C); Minimize light exposure

Overview SDS Protocol

Molecular weight

659.70

Our Buccutite™crosslinking technology provides the most convenient and effective crosslinking method to link two biomolecules with a high conjugation yield. Our method uses one pair of crosslinkers: Buccutite™ MTA and Buccutite™ FOL. MTA is added to one molecule, while FOL is added to another molecule. The cross-linking reaction is initiated by mixing Molecule-1-Buccutite ™ MTA and Molecule-2-Buccutite ™ FOL. This crosslinking reaction occurs under extremely mild and neutral conditions without any catalyst required. It is robust and efficient. Many of our customer have requested us to offer the stand-alone Buccutite™ MTA and Buccutite™ FOL reagents to expand the application of Buccutite™crosslinking technology. Protein-protein conjugations are commonly performed with a bifunctional linker such as SMCC. One end of the SMCC reacts (via NHS ester) with amines (-NH2) found in the amino acid lysine and N-terminus, and the other end reacts (via maleimide) with the thiol groups (-SH) found in the amino acid cysteine. However, SMCC-modified protein is extremely unstable and often self-reactive since proteins often contain both amine and thiol groups that cause significant amount of homo-crosslinking. In addition, it is quite difficult and tedious to quantify the number of maleimide groups on a protein.

Example protocol

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Buccutite™ MTA, NHS ester stock solution (20 mM)

Add 100 µL high-quality, anhydrous dimethylsulfoxide (DMSO) or dimethyl-formamide (DMF) to Buccutite™ MTA, NHS ester vial to prepare 20 mM stock solution.
Note Dissolve the dye immediately before starting the reaction.
Note The Buccutite™ MTA, NHS ester stock solution should be stored at -20 °C after preparation. Once reconstituted, the NHS ester reactive dye solution is not very stable, especially if exposed to moisture. It could hydrolyze into the nonreactive free acid in aqueous solutions.

SAMPLE EXPERIMENTAL PROTOCOL

Prepare protein working solution:For labeling 1 mg protein (assuming the concentrations are 10 mg/mL), prepare protein solution in pH=8.5 buffer by buffer exchange.

Note         The Buccutite™ MTA modification reaction is highly concentration dependent, the protein concentration should usually be 2-10 mg/mL. Concentrations lower than 2 mg/mL will greatly decrease the efficiency of the reaction.

Note         Protein solutions must be free of any amine-containing substances such as Tris, glycine, ammonium ions, or stabilizing proteins such as bovine serum albumin. You can dialyze protein against 10 mM phosphate-buffered saline (1x PBS), and then add 5% (v/v) of 1 M sodium bicarbonate buffer (pH 8.5–9.0) to antibody solution to adjust pH to ~8.5.

Note         The presence of low concentrations of sodium azide (<3 mM) or thimerosal (<1 mM) will not interfere with the conjugation reaction.
Add the amount of Buccutite™ MTA, NHS ester stock solution needed to the protein solution, mix well and incubate the reaction for 1 hour at room temperature with continuous stirring.

Note Please modify the dye-to-protein labeling ratio used in the reaction to achieve the desired number.
Purification the reaction mixture with desalting column. Purify the conjugate by column chromatography with PBS buffer or other buffer of choice.

Note If the reaction volume is ~100 uL, spin column (Cat# 60500) can be used to purify. If the reaction volume is > 1 mL, we recommend packing a column using Sephadex® G-25 media or Bio-Gel® P-6DG Gel with an appropriate volume to purify the reaction mixture.
Collect the purified protein/MTA solution.
Determine the protein concentration:Measuring absorbance at 280 nm is not an accurate way to determine protein concentration because MTA has strong absorbance at 280 nm. An easier way is to estimate the protein concentration by 85% yield. If you need more accurate protein concentration, BCA assay or other protein assay is compatible with MTA modified protein.

Determine the number of MTA groups:

The number of MTA groups can be determined with Buccutite™ MTA-Dye 650 (Cat#5370).

Table 1. Here is a reference for The number of MTA groups after Buccutite™ MTA modification reaction:

Antibody Concentration*	Labeling Ratio**	MTA numbers / IgG molecule
GXM IgG, 2 mg/ml	10:1	2.5~3.0
GXM IgG, 10 mg/ml	3:1	1.0~1.5
GXM IgG, 10 mg/ml	5:1	2.0~2.5
GXM IgG, 10 mg/ml	10:1	3.0~4.0

*: GxM IgG solution at specified concentration, pH~8.5, 100 µL /reaction

**: Molar ratio of Buccutite™ MTA, NHS and antibody during the reaction

Storage of Buccutite™ MTA modified protein:

Buccutite™ MTA modified protein conjugate can be stored in PBS at 2~8°C for at least 6 month without significant decrease of MTA number.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Buccutite™ MTA, NHS ester to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

	0.1 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	151.584 µL	757.92 µL	1.516 mL	7.579 mL	15.158 mL
5 mM	30.317 µL	151.584 µL	303.168 µL	1.516 mL	3.032 mL
10 mM	15.158 µL	75.792 µL	151.584 µL	757.92 µL	1.516 mL