Thermo Fisher Scientific EZ-Link TFP-PEG3-Biotin User guide

Brand: Thermo Fisher Scientific

Model: EZ-Link TFP-PEG3-Biotin

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

21219 EZ-Link TFP-PEG3-Biotin, 50mg

Molecular Weight: 694.74

NOOOH

HN NH

Spacer Arm: 32.6Å

Formula: C

Storage: Upon receipt store reagent at 4°C protected from moisture. Reagent is shipped at ambient

temperature.

Introduction

Thermo Scientific EZ-Link TFP-PEG3-Biotin is a long-chain, water-soluble, amine-reactive biotinylation reagent. This

reagent has a hydrophilic polyethylene glycol (PEG) spacer arm that imparts water solubility. This hydrophilic property is

transferred to the labeled macromolecule, which minimizes the possiblity of protein aggregation without affecting their

biological activities. The tetrafluorophenyl (TFP) functional group of this reagent reacts with primary and secondary amines.

Biotin is a small naturally occurring vitamin that binds with high affinity to avidin and streptavidin proteins. Because it is so

small (244Da), biotin can be conjugated to many proteins without altering their biological activities. Labeled proteins can be

purified from unlabeled proteins using streptavidin and avidin affinity resins (see Related Thermo Scientific Products), and

they can be detected easily in ELISA, dot blot or Western blot applications using streptavidin or avidin-conjugated probes.

Important Product Information

• TFP-PEG3-Biotin is moisture-sensitive. Store the vial of biotin reagent at 4°C with desiccant. To avoid moisture

condensation onto the product, equilibrate vial to room temperature before opening.

• As directed in the procedure, dissolve the biotin reagent immediately before use. The TFP moiety readily hydrolyzes and

becomes non-reactive; therefore, weigh and dissolve only a small amount of the reagent at a time, and do not prepare

stock solutions for storage. Discard any unused reconstituted reagent. The maximum solubility of TFP-PEG3-Biotin is

5-7mg/mL in water or buffer.

• Avoid buffers containing primary amines (e.g., Tris or glycine) as these will compete with the reaction. If necessary,

dialyze or otherwise desalt to exchange the protein sample into an amine-free buffer such as phosphate-buffered saline

(PBS; see Related Thermo Scientific Products).

• When biotinylating proteins in solution, excess non-reacted biotin is easily removed by size exclusion using either

desalting columns or dialysis. A 5 or 10mL desalting column is best suited for processing biotinylation reactions

involving 1-10mg of protein in approximately 0.5-2mL. For smaller amounts of protein and/or smaller reaction volumes,

both the biotinylation reaction and subsequent buffer exchange may be performed in a single Thermo Scientific Slide-A-

Lyzer MINI Dialysis Unit. For larger reaction volumes than can be processed with a desalting column, either split the

sample between two columns or use an appropriate Slide-A-Lyzer™ Dialysis Cassette.

0874.4

21219

EZ-Link™ TFP-PEG

-Biotin

Pierce Biotechnology

PO Box 117

(815) 968-0747

www.thermoscientific.com/pierce

3747 N. Meridian Road

Rockford, lL 61105 USA

(815) 968-7316 fax

General Procedure for Biotinylating Proteins

The degree of TFP-PEG3-Biotin incorporation can vary depending on the parameters of the biotinylation reaction, including

protein concentration, TFP-PEG3-Biotin concentration, pH and time. Commonly used reaction conditions for TFP-esters

include incubation temperatures at 4-37°C, pH values from 7 to 9, and incubation times ranging from a few minutes to

overnight.

A. Additional Materials Required

• Phosphate-buffered saline (PBS; e.g., Product No. 28372) or other amine-free buffer (pH 7-8) for use as reaction buffer

• Dimethylformamide (DMF, Product No. 20673)

• Desalting columns or dialysis units for buffer exchange (see Important Product Information and Related Thermo

Scientific Products)

B. Method

1. Dissolve 2mg of IgG in 1mL PBS.

2. Immediately before use, dissolve 1mg of TFP-PEG3-Biotin in 1mL of DMF. Add 20µL to the IgG solution.

Alternatively, prepare 1.4mM (1mg/mL) of TFP-PEG3-Biotin in PBS and immediately transfer 20µL to the IgG solution.

3. Incubate at room temperature for 1 hour or on ice for two hours.

4. Remove nonreacted biotin by dialysis or gel filtration.

5. Store the biotinylated protein at 4°C, or appropriate temperature, until ready for use.

Determination of Biotin Incorporation

Biotin incorporation can be estimated using the HABA [2-(4´-hydroxyazobenzene)-benzoic acid] method. In solution, the

HABA dye binds avidin, forming a complex with maximal absorption at 500nm. When biotin is added to the solution, its

higher affinity for avidin displaces the HABA and the absorption at 500nm decreases proportionately. The absorbance of the

HABA-avidin solution is measured before and after adding the biotin-containing sample. The change in absorbance relates to

the amount of biotin in the sample. The Thermo Scientific Pierce Biotin Quantitation Kit (Product No. 28005) contains a

biotinylated protein control and a premix of HABA and avidin supplied in convenient Thermo Scientific No-Weigh

Microtube packaging.

Related Thermo Scientific Products

69576 Slide-A-Lyzer MINI Dialysis Unit Kit

66382, 66807 Slide-A-Lyzer Dialysis Cassette Kits, 10K MWCO

89891, 89883 Zeba™ Spin Desalting Columns, 7K MWCO

28005 Pierce™ Biotin Quantitation Kit

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

Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This

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

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