Thermo Fisher Scientific Imject EDC Carrier Protein Spin Kits User guide

Brand: Thermo Fisher Scientific

Model: Imject EDC Carrier Protein Spin Kits

Type: User guide

INSTRUCTIONS

2187.0

77670 77671 77672

Imject® EDC Carrier Protein Spin Kits

Number Description

77670 Imject EDC Blue Carrier® Protein Spin Kit, 5 × 2 mg, carrier is supplied lyophilized; when

reconstituted with 0.2 ml of ultrapure water, buffer contains 0.05 M MES (2-[N-morpholino]-

ethanesulfonic acid), 0.15 M NaCl, 0.05 M sucrose; pH 6.5

77671 Imject EDC mcKLH Spin Kit, 5 × 2 mg, carrier is supplied lyophilized; when reconstituted with

0.2 ml of ultrapure water, buffer contains 0.05 M MES, 0.15 M NaCl, 0.05 M sucrose; pH 6.5

77672 Imject EDC BSA Spin Kit, 5 × 2 mg, carrier is supplied lyophilized; when reconstituted with 0.2 ml

of ultrapure water, buffer contains 0.05 M MES, 0.01 M sorbitol; pH 4.7

Kit Contents:

All kits contain sufficient materials for five conjugation reactions.

Imject EDC Conjugation Buffer, 30 ml, 0.1 M MES, 0.9 M NaCl, 0.02% sodium azide; pH 4.7

EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), 5 × 10 mg

Imject Purification Buffer Salts, 5 × 0.84 g, upon reconstitution this buffer contains 0.083 M sodium

phosphate, 0.9 M sodium chloride, 0.1 M sorbitol; pH 7.2

Zeba™ Spin Desalting Columns (7K MWCO), 5 × 2 ml, contains 0.05% sodium azide

Storage: Upon receipt store at 4°C. Product shipped on ice.

Introduction

The Thermo Scientific Imject EDC Carrier Protein Spin Kits enable effective one-step conjugation of a hapten to a carrier

protein using EDC as the crosslinker. The resulting conjugate is used for eliciting an immune response and antibody

production against the hapten. EDC reacts with exposed carboxyl and amino groups on peptides and proteins to form stable

bonds. These kits contain carrier proteins formulated in buffers compatible with EDC reactions and Thermo Scientific Zeba

Desalt Spin Columns, which offer exceptional desalting and protein recovery by centrifugation eliminating the waiting and

monitoring associated with gravity-flow columns.

Blue Carrier Protein is a hemocyanin from the Concholepas concholepas mollusk with immunogenic properties similar to

KLH but is more stable and efficient as a carrier protein for the production of antibodies to haptens and peptides. Mariculture

keyhole limpet hemocyanin (mcKLH) produces an excellent immune response and is currently the industry standard for

antibody production against a hapten or peptide. mcKLH is harvested from the hemolymph of giant keyhole limpets

(Megathura crenulata) maintained in environmentally optimized marine tanks and are not sacrificed during the hemolymph

collection. Bovine serum albumin (BSA) constitutes approximately half of the protein in plasma and is often used as a non-

relevant carrier in an ELISA to assess anti-peptide antibody titers.

Pierce Biotechnology PO Box 117 (815) 968-0747 www.thermo.com/pierce

3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax

Pierce Biotechnology PO Box 117 (815) 968-0747 www.thermo.com/pierce

3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax

Procedure for Hapten Conjugation using EDC

This protocol is designed to yield effective immunogens for a wide variety of haptens but is not necessarily optimal for a

specific hapten. Differences in size and structure of haptens will affect conjugation efficiencies. Using a molar excess of

hapten over carrier protein ensures efficient conjugation. Generally, a reaction with equal mass amounts of hapten and carrier

protein will achieve sufficient molar excess.

A. Conjugation Procedure

1. Reconstitute one vial of carrier protein (Blue Carrier Protein, mcKLH or BSA) by adding 200 μl of ultrapure water to

make a 10 mg/ml solution.

Note: Blue Carrier Protein and mcKLH form a suspension that typically appears translucent to whitish-blue. Do not

vortex or heat the suspension, which will precipitate the carrier.

2. Dissolve up to 2 mg of hapten in 450 μl of Imject EDC Conjugation Buffer.

Note: For haptens with limited solubility, use ≤ 30% DMSO in the final conjugation solution. Using a higher

concentration of DMSO might irreversibly denature the carrier protein.

3. Add the 450 μl of hapten solution to the 200 μl carrier protein solution.

4. For mcKLH and Blue Carrier Protein, dissolve one vial of EDC (10 mg) in 1 ml of ultrapure water and immediately add

50 μl of this solution to the carrier-hapten solution. For BSA, add the carrier-hapten solution to one vial of EDC (10 mg)

and dissolve by gentle mixing.

5. Incubate reaction for 2 hours at room temperature.

6. Purify the conjugate by desalting to remove non-reacted crosslinker and sodium azide.

B. Conjugate Purification by Desalting

• If the conjugate is to be used for injection within one week, PBS may be used for purification. If the conjugate will be

frozen, use the Imject Purification Buffer Salts for purification, which will preserve the product during freeze-thaw

cycles.

• If DMSO was used in the conjugation, prepare the Purification Buffer Salts with the same percentage of DMSO used for

the conjugation. This minimizes precipitation in the column during desalting.

• Desalting will not separate non-conjugated protein from conjugated protein; however, a large excess of hapten is used in

this protocol, making it unlikely that non-conjugated carrier exists in significant quantity.

• If a precipitate formed during conjugation, centrifuge the precipitated material, collect the supernatant and save the

precipitate. Purify only the supernatant. Combine the purified conjugate to the precipitate.

1. Dissolve the contents of one bottle of Purification Buffer Salts by adding 10 ml degassed, ultrapure water to the bottle.

Excess buffer can be stored at 4ºC for one week.

2. Twist off the bottom closure of the desalting column and loosen the cap. Place the column in a collection tube.

3. Centrifuge column at 1,000 × g for 2 minutes to remove storage solution. When using fixed-angle rotors, place a mark on

the side of the column where the compacted resin is slanted upward. Place column in centrifuge with the mark facing

outward in all subsequent centrifugation steps.

4. Remove the cap and slowly add 1 ml of purification buffer to the column. Centrifuge at 1,000 × g for 2 minutes to

remove buffer. Repeat this step three additional times, discarding the buffer from the collection tube.

5. Place the column in a new collection tube and slowly apply the sample to the center of the compact resin bed.

6. Centrifuge at 1,000 × g for 2 minutes to collect the sample.

7. (Optional) After the conjugate-containing fraction is collected, the non-conjugated hapten can be recovered by

continuing to add buffer to the column and collecting additional fractions.

8. The hapten-carrier conjugate can now be used for immunization. If the hapten-carrier conjugate is to be stored for more

than a few days, sterile filter the conjugate and store in a sterile container at 4ºC or -20ºC.

Pierce Biotechnology PO Box 117 (815) 968-0747 www.thermo.com/pierce

3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax

Troubleshooting

Problem Possible Cause Solution

Peptide not readily soluble in aqueous

buffer

Add up to 30% DMSO in the final reaction Hapten-carrier conjugate

precipitates

Incorrect amount of DMSO in reaction

solution

Use ≤ 30% DMSO in the final reaction or

the carrier protein may denature and

precipitate

Hapten-carrier conjugate

precipitates in desalt column

DMSO added to reaction but not added

to desalt buffer

Add DMSO to the purification/desalt buffer

at the same concentration as in the reaction

buffer

Make sure the centrifuge is working

properly

Sample or buffer does not flow

through the desalt resin

Centrifugation problem

Remove the column’s bottom closure

No stacker used Apply a stacker above sample Low conjugate recovery

Unstable protein conjugate Equilibrate column in a suitable buffer

Related Thermo Scientific Products

77171 Imject BSA (in MES Buffer), 2 mg

77140 Imject Freund’s Complete Adjuvant, 5 × 10 ml

77145 Imject Freund’s Incomplete Adjuvant, 5 × 10 ml

77159 Imject Purification Buffer Salts, 5 g

77161 Imject Alum, 50 ml

77162 Imject EDC Conjugation Buffer, 30 ml

89889 Zeba Spin Desalting Columns, 7K MWCO, 5 × 2 ml

Blue Carrier is a registered trademark of Biosonda S.A.

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)

use of the Products in a manner for which they were not designed, or (iv) improper storage and handling of the Products.

Current versions of product instructions are available at www.thermo.com/pierce. For a faxed copy, call 800-874-3723 or contact your local distributor.

subsidiaries. Printed in the USA.