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Buccutite™ MTA, NHS ester
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
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.
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
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
| 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 |
Molarity calculator
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Safety data sheet