Thermo Fisher Scientific NHS-AMCA Sulfo-NHS-AMCA User guide

Brand: Thermo Fisher Scientific

Model: NHS-AMCA Sulfo-NHS-AMCA

Type: User guide

INSTRUCTIONS

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

Number Description

33005

NHS-AMCA, 5mg

Molecular Weight: 330

Storage: Upon receipt store product at 4°C. Product is shipped at ambient temperature.

33010

Sulfo-NHS-AMCA, 5mg

NH2

-O

Na+

Molecular Weight: 431

Storage: Upon receipt store product at -20°C. Product is shipped at ambient temperature.

AMCA Excitation/Emission Wavelengths: 345-350/440-460nm (blue)

Introduction

The Thermo Scientific NHS-AMCA (succinimidyl-7-amino-4-methylcoumarin-3-acetate) and Sulfo-NHS-AMCA

(sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetate) are amine-reactive fluorophores for labeling antibodies, proteins

and other compounds. These fluorophores each contain an N-hydroxysuccinimide (NHS) ester, which is the simplest and

most commonly used reactive group for labeling proteins. The NHS ester on the dye cross-links to primary amines on

proteins, resulting in the formation of a stable, covalent amide bond and release of the NHS group. AMCA emits a blue

fluorescence and is well suited for double and triple labeling with fluorochromes such as fluorescein, lissamine and others

that are red or green.

Important Product Information

• NHS ester-activated fluors are moisture-sensitive. To avoid moisture condensation onto the product and subsequent

inactivation of the NHS ester, vial must be equilibrated to room temperature before opening.

• Prepare these labeling reagents immediately before use. Do not store NHS-ester reagents in aqueous solutions.

Procedure for Protein Labeling

The following is an example application for this product. Specific applications require optimization.

A. Protein Preparation

The optimal labeling buffer is 0.05M sodium borate buffer at pH 8.5 (Thermo Scientific BupH Borate Buffer Packs,

Product No. 28384). Buffers that contain primary amines (e.g., Tris or glycine) are not compatible because they will react

with the NHS-ester moiety. Dissolve protein directly in the 0.05M sodium borate buffer. For each labeling reaction, use 50µL

to 1mL of purified protein sample at 1-10mg/mL. After reconstitution, proceed to the Labeling Calculations Section. If the

protein is already in a buffer, perform a buffer exchange into the borate buffer by dialysis or gel filtration.

Note: The following buffers may be substituted for borate buffer: 0.1M sodium phosphate, 0.15M NaCl at pH 7.2-7.5 (e.g.,

BupH™ Phosphate Buffered Saline Packs, Product No. 28372) or 0.1M sodium carbonate at pH 8.3-9.0.

0087.2

33005 33010

NHS-AMCA

Sulfo-NHS-AMCA

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

B. Labeling Calculations

The amount of fluorescent-labeling reagent to use for each reaction depends on the amount of protein to be labeled. By using

the appropriate molar ratio of labeling reagent to protein, the extent of conjugation can be controlled. Use a 10-fold molar

excess of the dye when the protein concentration is 2-5mg/mL. For protein concentrations < 2mg/mL, use 15-fold molar

excess of dye to protein. For protein concentrations > 5mg/mL, use eight-fold molar excess. Determine the amount of AMCA

solution to use before beginning the labeling reaction.

1. Calculate amount (mg) of AMCA to be added to the labeling reaction:

AMCA of mg _______ AMCA ofMW 10

protein ofMW (mg)protein ofamount =××

2. Calculate microliters of AMCA solution to add to the reaction:

3mg/mLat solution AMCA μL _______

3mg

1000µL

AMCA of mg =×

• 10 = Molar ratio excess of the AMCA to protein

• 1000µL = Solvent volume in which the 3mg of AMCA is dissolved.

C. Labeling Reaction

Note: To protect reagent from moisture, allow AMCA to equilibrate to room temperature before opening the vial.

1. Transfer the protein solution to a reaction tube.

2. Dissolve 3.0mg NHS-AMCA in 1mL of DMSO or 3.0mg of Sulfo-NHS-AMCA in 1mL of ultrapure water.

3. Transfer the appropriate amount of dye (based on calculations) to the reaction tube containing the protein. Mix well and

incubate at room temperature for 1 hour.

4. Remove non-reacted dye from the protein by desalting or dialysis.

5. Store labeled protein protected from light at 4°C for up to one month. Alternatively, store labeled protein in single-use

aliquots at -20°C.

D. Calculate the Degree of Labeling

The non-reacted dye must be completely removed for optimal results and accurate determination of the fluor-to-protein ratio.

1. Use a 1cm path length cuvette to measure the absorbance of the labeled protein at 280nm and 346nm. It may be

necessary to dilute a small amount of the desalted/dialyzed sample.

2. Calculate the concentration of the protein in the sample:

• ε = protein molar extinction coefficient (e.g., the molar extinction coefficient of IgG is ~210,000 M-1 cm-1)

• Amax = A346

• CF = Correction factor

max

280

A A

; CF for AMCA = 0.19

factordilution

CF) (A -A

(M)ion concentratProtein max280 ×

3. Calculate the degree of labeling:

• ε´ = molar extinction coefficient of the fluorescent dye = 19,000 M-1 cm-1 for AMCA

factordilution

(M)ion concentratprotein ε´protein labeled theof A

protein moleper fluor Moles max ×

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

Troubleshooting

Problem

Cause

Solution

The application in which the

dye-labeled protein was used

was unsuccessful

The protein was not labeled Before troubleshooting, determine if the protein

is labeled by calculating the degree of labeling

after thorough desalting or dialysis.

Note: For fluor-labeled antibodies, the degree

of labeling should be > 1.

The protein was not labeled Conjugation buffer contained primary

amines that interfered with the reaction Use a conjugation buffer free of primary

amines such as Tris or glycine.

The NHS ester has hydrolyzed and is

non-reactive Prepare labeling reagent immediately before

use. Do not store NHS-ester reagents in

solution.

Additional Information on Our Website

• Tech Tip #43: Protein stability and storage

• Tech Tip #31: Calculate dye/protein (F/P) molar ratios

• Tech Tip #20: Dialysis: an overview

• Tech Tip #6: Extinction coefficients guide

• Tech Tip #3: Determine reactivity of NHS ester biotinylation and crosslinking reagents

• Tech Tip #30: Modify and label oligonucleotide 5´ phosphate groups

Related Thermo Scientific Products

Visit our website for a complete list of DyLight Products, including chemically reactive dyes, dye-protein conjugates

and labeling kits.

62262 DyLight 550 NHS Ester, 1mg

62265 DyLight 650 NHS Ester, 1mg

62290 DyLight 550 Maleimide, 1mg

62295 DyLight 650 Maleimide, 1mg

20673 Dimethylformamide (DMF), 50mL

28384 BupH Borate Buffer Packs, 40 packs

28372 BupH Phosphate Buffered Saline Packs, 40 packs

43230 Dextran Desalting Columns, 5K MWCO, 5mL, 5/pkg

69550 Slide-A-Lyzer® MINI Dialysis Units, 3.5K MWCO, 50/pkg

89890 Zeba™ Spin Desalting Columns, 7K MWCO, 2mL, 25/pkg

89892 Zeba Spin Desalting Columns, 7K MWCO, 5mL, 25/pkg

89894 Zeba Spin Desalting Columns, 7K, MWCO, 10mL, 25/pkg

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

This product (“Product”) is warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale,

as set forth in the Product documentation, specifications and/or accompanying package inserts (“Documentation”) and to be free from defects in material and

workmanship. Unless otherwise expressly authorized in writing, Products are supplied for research use only. No claim of suitability for use in applications

regulated by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the

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warranty does not extend to anyone other than the original purchaser of the Product (“Buyer”).

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