Enabling Epigenetics Research

Sortag-IT™ 3X DBCO Click Labeling Kit
Catalog No. 13116(Version A1)

Copyright 2023 Active Motif, Inc.

Information in this manual is subject to change without notice and does not constitute acommitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any
warranty of any kind, either explicit or implied. Information may be changed or updated inthis manual at any time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated,
in whole or in part, without the prior written consent of Active Motif, Inc. This documentation
is proprietary information and protected by the copyright laws of the United States andinternational treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2023 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All
rights reserved.
All trademarks, trade names, service marks or logos referenced herein belong to theirrespective companies.
For research use only. Not for use in diagnostic procedures.

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Contents
Overview  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Process Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  2
Kit Components and Storage  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4Sortag-IT™ 3X DBCO Click Labeling Kit - Click Protocol  . . . . . . . . . . . . . . . . . . . . . .  5Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  7
Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  8
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  9

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Overview
The Sortag-IT™ Labeling Kits* are designed to label Active Motif’s highly specific AbFlex®
recombinant antibodies (rAb) via the Sortase tag recognition sequence (LPXTG) that isincorporated into the heavy chains of each AbFlex® antibody. Sortase A belongs to thesortase family of transpeptidases found in Gram-positive bacteria and is used to catalyze theattachment of poly-Glycine containing labels to the recognition sequence. The Sortag-IT™
Labeling Kit uses Active Motif’s Sortase A5 pentamutant which has activity >15 times wildtype Sortase, allowing for a faster, more efficient labeling reaction. Each antibody contains
two Sortase tag sequences.
The Sortag-IT™ conjugation system is site directed, preventing non-specific conjugationthat might interfere with antibody binding and provides control over how many conjugatesare attached per antibody. Active Motif’s latest advance in sortase mediated conjugation
incorporates click chemistry, resulting in simplified conjugations and more conjugates
per antibody.
ProductFormat

Catalog No.

Sortag-IT™ 3X DBCO Click Labeling Kit

3 x 100 µg

13115*US Patent 9,267,127

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Process Overview

PEG

PEG

PEG

Reaction

(G)n

PEG

PEG

PEG

R1(G)nClickChemistry

NN NNN NN3

R1

BiomoleculeAzide

DBCOConjugate

R1

N
N N

Figure 1: Click Chemistry Reaction Schematic.
Click chemistry works through the presence of an Azide group on the donor molecule and aDBCO group on the acceptor molecule. Covalent attachment is achieved by simply mixing thetwo components.

The new Sortag-IT™ linkers provide even more control over the number of conjugates added
per antibody and are compatible with any number of commercially available click chemistry
conjugates such as dyes or oligos. Click chemistry reactions are fast, simple to use, versatile,and give high product yields. First, Sortase is used to attach the linker to the Sortase
recognition sequence in the AbFlex® antibody (Figure 2). Then click chemistry can be usedto add Azide containing molecules to the AbFlex® - 3X DBCO acceptor molecule (Figure 3).
Labeling of both heavy chains using this linker results in 2 conjugation sites per antibody.
12

34(G)n—
Sortase A5
LPXTG——LPXTG—LPXTG(G)n—(G)n—Sortase A5

Sortase A5—LPXT

Sortase A5—LPXT(G)n—GDBCO
moiety(G)n—*=(G)nPEG

PEGPEG

DBCO
moietyDBCO
moietyFigure 2: Sortag-IT™ 3X DBCO Click Labeling Kit and Sortase A5 Labeling Scheme.
(1) Combine the buffer exchanged AbFlex® recombinant antibody (rAb) of interest in Reaction Bufferwith the (Gly)5- 3X DBCO Click label at a ratio of greater than or equal to 5 nmol label to 1 nmol
antibody in the presence of Sortase A5 enzyme. (2) Sortase A5 will bring the (Gly)5- 3X DBCO
Click label to the Sortase recognition sequence (LPXTG) on the antibody. (3) Sortase A5 will cleave
the bond between the Threonine and the Glycine of the LPXTG recognition sequence creating anacyl enzyme intermediate which allows for the attachment of the (Gly)5- 3X DBCO Click label. (4)Following the reaction, the labeled AbFlex® antibody can be purified away from the free label andthe Sortase A5 enzyme inactivated with Stop Solution. This results in an antibody containing a 3X
DBCO group for use in click chemistry.
*This kit provides a poly-Glycine label with three dibenzocyclooctyne (DBCO) moieties as click chemistry component ((Gly)5- 3X DBCOClick label).

activemotif.com|2Azide/DBCO
labeled AbFlex®
Antibody

LPXT(G)nDBCO/Azide(click moiety)

Conjugate

Oligo(barcode)

ProteinRadiolabel

Beads

Fluorophore

Carbohydrate

PhysiologicalConditions
LPXT(G)n= Click chemistry moieties
Figure 3: The Sortag-IT™ 3X DBCO Click Labeled AbFlex® Antibody Used in Click
Chemistry Reactions.
The labeled AbFlex® antibody containing a 3X DBCO click chemistry groups at the C-terminus
can be reacted with a series of Azide containing molecules resulting in a click chemistry
mediated conjugation.

activemotif.com|3Kit Components and StoragePlease store each component at the temperature indicated in the table below. Each
Sortag-IT™ 3X DBCO Labeling Kit provides sufficient materials to label 3 x 100 µg AbFlex®
recombinant antibody with a yield > 60%.
Kit Component

Quantity

Storage

Sortase A5 enzyme

12 units (3 tubes)-80°C

Reaction Buffer AM3

3.2 mL (3 tubes)

4°C(Gly)5- 3X DBCO Click label

24 µL

-20°C

Stop Solution AM38 µL

RT

Purification columns

6 columns (3 per bag)

RT

Additional Materials Required• 100 µg AbFlex® Recombinant antibody per reaction (for a complete product listing
visit activemotif.com/abflex)• 1.5 mL microcentrifuge tubes and microcentrifuge• Thermomixer, or equivalent instrument capable of incubating samples at 30°Cwith shaking• PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4)

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Sortag-IT™ 3X DBCO Click Labeling Kit - Click Protocol
Buffer Exchange1.

Add 250 µL of Reaction Buffer AM3 to the AbFlex® antibody tube and transfer to theincluded column.

AbFlex®
+AM3 BufferColumn

2.

Spin the column at 13,500 x g for 5 minutes in a microcentrifuge. Discard flow throughand replace column in the collection tube.

3.

Add 400 µL of Reaction Buffer AM3 to the column/collection tube assembly and spin
the column at 13,500 x g for 5 minutes. Discard flow through.

4.

Add 150 µL Reaction Buffer AM3 to the column. Place the column inverted into a new
microcentrifuge tube.

5.

Spin column at 800 x g for 2 minutes. Save the flow through and discard the column.
The flow through contains your buffer exchanged antibody.

6.

Measure the volume and add Reaction Buffer AM3 to achieve a total of 250 µL.

Labeling
7.

Thaw the vial of Sortase A5 enzyme on ice.

8.

Set up the labeling reaction by adding the reagents in the order shown in the tablebelow and mix by pipetting.
Kit Component
AbFlex® antibody in Reaction Buffer AM3

9.

Volume to add
250 µL(Gly)5- 3X DBCO Click label (5 mM)

8 µL

Sortase A5 enzyme (1 unit/µL)

4 µL

Incubate the labeling reaction at 30°C for 1 hour with shaking at 1000 rpm using aThermomixer or similar device.

activemotif.com|5Label Clean-Up
10. Following the incubation, remove excess label from the reaction by purifying with theincluded column.
a. Add 200 µL of PBS to the antibody labeling reaction.
b. Add the entire reaction to the column.
c. Spin column at 13,500 x g for 5 minutes in a microcentrifuge. Discard flow throughand replace the column in a new collection tube.
d. Wash the column by adding 400 µL PBS then spin column at 13,500 x g for 5
minutes in a microcentrifuge. Discard the flow through and replace the column inthe collection tube.
e. Repeat the wash from step d for two additional times for a total of three washes.
f. Add 100 µL PBS to the column. Place the column inverted into a new
microcentrifuge tube.
g. Spin column at 800 x g for 2 minutes to collect the labeled antibody.
11. Add 2.5 µL Stop Solution AM3 to labeled antibody to inactivate the Sortase A5
enzyme. Invert to mix.
12. Use immediately for click conjugation, or store at 4°C for up to 1 month.
Sortase Site Directed Click Chemistry Reaction (not included in the kit):
13. Following the lab clean-up proceed to use the 3X DBCO labeled AbFlex® antibody forclick chemistry.
14. Set up the click chemistry reaction by adding 400 pmol of Azide biomolecule to 20
pmol of 3X DBCO labeled AbFlex® antibody (labeled in this kit).
15. Incubate the reaction at 4°C for 24 hours then remove excess label using size
exclusion or purification of choice.

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6

Appendix
Application Data

Figure 4: AbFlex® CTCF Antibody (rAb) Conjugated to DNA Using Active Motif’s 3X DBCO
Click LabelAbFlex® CTCF antibody (rAb) (Cat No. No 91285) was labeled with Sortag-IT™ 3X DBCO ClickLabeling Kit (Cat No. No 13116). Then 20 pmol of 3X DBCO labeled antibody was mixed with 400
pmol of 70-mer oligonucleotide bearing an Azide* group and incubated at 4°C overnight. The
samples were loaded onto a 4-12% SDS-PAGE and stained with Coomassie blue.
Lane 1: AbFlex® CTCF unlabeled control antibody.
Lane 2: AbFlex® CTCF antibody (rAb) 3X DBCO labeled and conjugated to a 70- oligonucleotide.
The red arrow shows the antibody with 1 oligonucleotide conjugated.
The blue arrow shows the antibody with 2 oligonucleotides conjugated.
The green arrow shows the antibody with 3 oligonucleotides conjugated.
The yellow arroe shows the antibody with 4 oligonucleotides conjugated.

*Azide-modified oligonucleotide was prepared from a commercial amine-modified oligonucleotide. Commercial amine-modified (3’ or 5’)
oligonucleotide was dissolved in water (0.3-0.5 mM; 60 µL) and precipitated in pre-chilled (-20ºC) 3% lithium perchlorate in acetone (1.7mL). The pellet was thenwashed with acetone (2 x 0.7 mL) and dried. The oligonucleotide pellet was dissolved in 60 µL 0.1M MOPS buffer(pH 7.7). Azido-PEG-NHS (9 µL; 25 mg/mL in DMF) was added and the reaction mixture was incubated for 2 hr followed by precipitation in
pre-chilled (-20ºC) 3% lithium perchlorate in acetone (1.7 mL). The pellet was washed with acetone (2 x 0.7 mL) then dried. Azide modified
oligonucleotide was used for the conjugation without additional purification.
Azido-PEG4-NHS Ester (Catalog No AZ103, Click Chemistry Tools)
Alternatively, oligonucleotides can be purchased directly from vendors such as IDT with 3’ or 5’ Azide modifications.

activemotif.com|7Troubleshooting Guide
Problem/Question

Recommendation

How can I determine thelabeling efficiency?

The maximum number of labels that can be added to an AbFlex®
antibody is 2 (this is a theoretical maximum as steric hindrance
and reaction yield may prohibit the addition of 2 labels per
antibody molecule).
We recommend performing a click reaction with Azide-modified
oligonucleotide (40-70-mer) followed by SDS-PAGE as described inApplication Data section.
A commercially available dye modified with Azide can be used. Afterperforming the “click” reaction, dye/protein ratio can be determinedaccording to the dye manufacturer protocol.

Can I label for shorter or longer
time periods?

The Sortag-IT Labeling Kits have been optimized for the most efficientlabeling time based on the use of 100 µg of antibody with therecommended amount of poly-Glycine label per reaction. Altering thelabeling time may result in decreased labeling efficiency.

Can I label smaller amountsof antibody?

Yes, smaller amounts of antibody (25 - 100 µg) can be labeled with
the Sortag-IT Labeling Kits, but the amount of Sortase A5 enzyme
and poly-Glycine label should not be modified in the reaction. Volume
differences should be corrected using Reaction Buffer AM3 to maintaina total volume of 262 µL per reaction. Optimization of labeling time may
be required. Please note that the yield for smaller antibody amounts
may be diminished.

Do I need to purify mylabeled antibody?

It is strongly recommended to purify the labeled antibody away from theexcess label and Sortase A5 prior to use. If the antibody will be used
immediately, skipping purification may be acceptable. However, storageof unpurified antibody over time will result in removal of the label. Any
active Sortase A5 will continue to cleave at the recognition sequenceand may create large antibody complexes or completely remove
the poly-Glycine labels resulting in unlabeled antibody. Therefore,
purification prior to use and storage is strongly recommended.

activemotif.com|8Technical Services

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If you need assistance at any time, please call or send an e-mail to Active Motif Technical
Service at one of the locations listed below.

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