Thermo Fisher Scientific Pierce Maleimide Activated 96-Well Plates User guide

Brand: Thermo Fisher Scientific

Model: Pierce Maleimide Activated 96-Well Plates

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

15150 Pierce Maleimide Activated Plates (8-well strips), 5 each

15152 Pierce Maleimide Activated Plates (white, 96-well), 5 each

15153 Pierce Maleimide Activated Plates (black, 96-well), 5 each

Binding Capacity: 100-150pmol of a sulfhydryl-containing peptide (307Da)/well

Activation Level: 100µL

Note: Plates are supplied pre-blocked with bovine serum albumin.

Storage: Upon receipt store plates at 4°C protected from moisture. Once opened, place unused plates

in a resealable bag with desiccant and store at 4°C. Plates are shipped at ambient temperature.

Introduction

The Thermo Scientific Pierce Maleimide Activated Plates are ideal for binding sulfhydryl-containing molecules that are

difficult to coat onto polystyrene plates, such as peptides that contain a terminal cysteine. These coated plates are an

especially useful tool for assessing specific anti-hapten antibody titers during antibody production. Plates are available in

clear for colorimetric assays, white for chemiluminescent assays, and black for fluorescent assays.

Important Product Information

• Maleimides react with free sulfhydryl group(s) at pH of 6.5-7.5, forming stable thioether linkages. Reaction with amines

becomes significant at pH > 7.5.

• Some sulfhydryl-containing peptides and proteins may oxidize in solution and form disulfide bonds, which cannot react

with maleimides. Disulfide bonds can be reduced to produce free sulfhydryls. The Thermo Scientific Pierce Immobilized

Reductant Column (Product No. 77701) and Immobilized TCEP Disulfide Reducing Gel (Product No. 77712) enable

peptide or protein reduction while recovering the sample in the absence of reducing agents.

• Sulfhydryls can be introduced via amine modification using N-succinimidyl S-acetylthioacetate (SATA, Product No.

26102) or 2-iminothiolane•HCl (Traut’s Reagent, Product No. 26101).

General ELISA Procedure

Note: To determine sulfhydryl content of the sample, perform an Ellman’s assay (Ellman’s Reagent, Product No. 22582).

A. Materials Required

• Binding Buffer: 0.1M sodium phosphate, 0.15M sodium chloride, 10mM EDTA; pH 7.2

• Wash Buffer: 0.1M sodium phosphate, 0.15M sodium chloride, 0.05% Tween®-20 Detergent; pH 7.2

• Cysteine•HCl (Product No. 44889)

• Primary antibody

• Enzyme-conjugated secondary antibody

• Enzyme substrate (e.g., for HRP use the Thermo Scientific TMB Substrate Kit, Product No. 34021)

0702.4

15150 15152 15153

Pierce® Maleimide Activated 96-Well Plates

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. Method

1. Wash plate wells three times with 200µL each of Wash Buffer.

2. Prepare the sulfhydryl-containing peptide at 1-50µg/mL in Binding Buffer.

3. Add 100-150µL of the peptide solution to each well and incubate for ≥ 2 hours at room temperature or overnight at 4°C.

For best results, use a plate mixer during incubation.

4. Wash wells three times using 200µL of Wash Buffer for each wash.

5. Immediately before use, prepare cysteine solution at 10µg/mL. Add 200µL and incubate for 1 hour at room temperature

to inactivate excess maleimide groups.

6. Wash wells three times using 200µL of Wash Buffer for each wash.

7. Add 100µL of primary antibody per well and incubate for 1 hour at room temperature.

8. Wash wells three times using 200µL of Wash Buffer for each wash.

9. Add 100µL per well of secondary antibody. Incubate for 1 hour at room temperature.

10. Wash wells three times using 200µL of Wash Buffer for each wash.

11. Add the enzyme substrate and develop according to manufacturer’s recommendations.

Related Thermo Scientific Products

37070 SuperSignal® ELISA Pico Chemiluminescent Substrate, 100mL, peroxidase substrate

15169 QuantaBlu™ Fluorogenic Peroxidase Substrate Kit

34028 1-Step Ultra TMB-ELISA, 250mL

37615 1-Step ABTS Substrate, 250mL

31430 Goat-anti-Mouse HRP Conjugate, 2mL

31460 Goat-anti-Rabbit HRP Conjugate, 2mL

28372 BupH™ Phosphate Buffered Saline Packs, 40 packs

21224 Streptavidin, Horseradish Peroxidase Conjugated, 2mg

31001 NeutrAvidin® Protein, Horseradish Peroxidase Conjugated, 2mg

77666 Imject® Maleimide-Activated mcKLH Spin Kit

General Reference

Smyth, D.G. et al. (1964). Reaction of N-ethylmaleimide with peptides and amino acids. Biochem J 91:589.

Tween is a trademark of Croda International Plc.

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”).

No other warranties, express or implied, are granted, including without limitation, implied warranties of merchantability, fitness for any particular

purpose, or non infringement. Buyer’s exclusive remedy for non-conforming Products during the warranty period is limited to replacement of or

refund for the non-conforming Product(s).

There is no obligation to replace Products as the result of (i) accident, disaster or event of force majeure, (ii) misuse, fault or negligence of or by Buyer, (iii)