Sortag-IT™ HRV 3C-Biotin Labeling Kit
Catalog No.: 13111
Format:  3 x 100 µg
Description
The Sortag-IT™ Biotin Labeling Kits* are designed to label Active Motif’s highly specific AbFlex™ recombinant antibodies (rAb) via theSortase tag recognition sequence (LPXTG) that is incorporated 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 the attachment of poly-Glycine containinglabels to the recognition sequence. The Sortag-IT Labeling Kit uses Active Motif’s Sortase A5 pentamutant sortase which has activity>15 times wild-type Sortase, allowing for a faster, more efficient labeling reaction. Each antibody contains two Sortase tag sequences
and can add a maximum of two labels per antibody. Simply combine your AbFlex antibody with the poly-Glycine label, add Sortase A5,
and incubate for 1 hour at 30°C. Purification columns are included to remove excess label and Stop Solution is provided to inactivate theSortase A5 enzyme. Labeled antibodies are ready for downstream analysis or can be stored at 4°C for up to 3 months.

Figure 1:  The Sortag-IT™ antibody labeling system.
(1) Combine the AbFlex™ recombinant antibody (rAb) of interest with the desired poly-Glycine (G)n label at a ratio of greater than or equal to 5 nmol label to 1 nmol antibody in the presence of Sortase A5
enzyme and reaction buffer. (2) Sortase A5 will bring poly-Glycine label to the Sortase recognition sequence (LPXTG) on the antibody. (3) Sortase A5 will cleave the bond between the Threonine and theGlycine of the LPXTG recognition sequence creating an acyl enzyme intermediate which allows for the attachment of the poly-Glycine label. (4) Following the reaction, the labeled AbFlex antibody can bepurified away from the free label and the Sortase A5 enzyme inactivated with Stop Solution.

The HRV 3C-Biotin label is designed to provide a cleavable release of the AbFlex antibody from biotin using the Rhinovirus (HRV) protease. This labeling strategy enables antibody capture using biotin/streptavidin followed by release of the antibody via HRV 3C cleavage.
Note:

When performing a HRV 3C cleaving following antibody labeling, it is important to use a reaction buffer which does notcontain DTT, which can reduce disulfide bonds in the antibody heavy and light chains. For your convenience, the Sortag-IT
HRV 3C-Biotin labeling kit includes reaction buffer suitable for this cleavage.

Contents
Each Sortag-IT™ HRV 3C-Biotin Labeling Kit provides sufficient materials to label 3 x 100 µg AbFlex™ recombinant antibody with a yield >50%.
• 3 units Sortase A5 enzyme; Store at -80°C• 1 ml Reaction Buffer AM3; Store at 4°C• 5 mM (Gly)5- HRV 3C-Biotin label; Store at -20°C• 10 µl Stop Solution AM3; Store at RT• 3 ea Purification columns; Store at RT• 15 µl HRV 3C Protease; Store at -20°C• 10X HRV 3C Reaction Buffer; Store at RTItems Required but not included• 100 µg AbFlex™ Recombinant antibody per reaction (see www.activemotif.com/abflex for a complete product listing)• 1.5 ml microcentrifuge tubes and microcentrifuge• Thermomixer, or equivalent instrument capable of incubating samples at 30°C with shaking• PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4* US Patent 9,267,127

North America 877 222 9543

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

www.activemotif.com

Procedure for Sortag-IT HRV 3C-Biotin:
Reaction conditions are label-specific.
1. Thaw the vial of Sortase A5 enzyme on ice.
2. Set up the labeling reactions in a 1.5 ml microcentrifuge tube. Add reagents in the order shown below and mix by pipetting.
Reagent

Volume to add

AbFlex antibody (1 µg/µl)

100 µlReaction Buffer AM394 µl(Gly)5-HRV 3C-Biotin label (5 mM)

5 µl

Sortase A5 enzyme (1 unit/µl)

1 µl

Total Volume

200 µl3. Incubate the labeling reactions at 30°C for 1 hour with shaking at 1000 rpm.
4. Following the incubation, remove excess label from the reaction by purification using the included columns.
a. Equilibrate each column by adding 450 µl PBS.
b. Spin columns at 13,500 x g for 5 minutes in a microcentrifuge. Discard flow through and replace column in the collection tube.
c. Add 250 µl PBS to each antibody labeling reaction (450 µl final volume).
d. Add diluted labeling reactions to the equilibrated column.
e. Spin columns at 13,500 x g for 5 minutes in a microcentrifuge. Discard flow through and replace column in the collection tube.
f. Add 450 µl PBS to wash the column. Spin columns at 13,500 x g for 5 minutes in a microcentrifuge. Discard flow through andreplace column in the collection tube.
g. Repeat the wash from step f two additional times for a total of three washes.
h. Add 100 µl PBS to the column. Place the column inverted into a new microcentrifuge tube.
i. Spin columns at 800 x g for 2 minutes to collect labeled antibody.
5. Add 2.5 µl Stop Solution AM3 to each labeled antibody to inactivate the Sortase A5 enzyme.
6. Use immediately, or store at 4°C for up to 3 months.
Procedure for Sortag-IT HRV 3C-Biotin Cleavage:
Cleave 5 - 100 µg  of HRV 3C-Biotin labeled antibody using 4 Units (U) of HRV 3C Protease. An example of a reaction is shown below. Scaleaccordingly.
Reagent

Volume to add

AbFlex antibody (5 - 100 µg)

Reaction-specific

10X HRV 3C Reaction Buffer

5 µl

HRV 3C Protease (2 U/µl)

2 µl

H20Adjust volume to 50 µl Total VolumeTotal VolumeNorth America 877 222 9543

Europe +32 (0)2 653 0001

50 µl

Japan +81 (0)3 5225 3638

www.activemotif.com

Appendix
Application Data

Dot blot of AbFlex RNA pol II CTD Ser2 and AbFlex RNA pol II CTD Ser5 conjugated antibodies
The Sortag-ITTM HRV-3C Biotin Labeling Kit or Sortag-ITTM Biotin Labeling  Kit (Cat. No. 13105) were used to directly conjugate 100 µg of Active Motif’s AbFlex® RNA pol II CTD phospho Ser2 and AbFlex® RNApol II CTD phos Ser5 antibodies with Biotin or HRV 3C Biotin. Following purification steps, 0.5 µg or each antibody was spotted onto nitrocellulose membrane and blocked with TBST containing 5% milk. Theblot was then probed using Streptavidin-HRP (BioLegend, Cat. No. 405210) for 1 hour at room temperature. The blot was washed and developed using the Bio-Rad Clarity kit. Results show the detection ofbiotin labeled antibodies by Streptavidin-HRP

Western blot analysis of HRV 3C Protease cleavage. of Histone H3K9ac-TEV-Biotin directly conjugated antibody.
First, 100 µg AbFlex® RNA pol II CTD phoshpo Ser2 recombinant antibody (Catalog No. 91115) and 100 µg AbFlex® RNA pol II CTD phospho Ser5 recombinant antibody (Catalog No. 91119) were labeled with
HRV 3C Biotin using the Sortag-IT HRV 3C Biotin Labeling Kit. Following purification, 5 µg of each labeled antibody and 5 µg of the equivalent antibody labeled with a non-cleavable biotin were treated with
4 units of HRV 3C protease for 2 hours at 4˚C. After cleavage, 16 µl of each antibody was run on a gel and transferred to a nitrocellulose membrane. The blot was then incubated with a streptavidin-HRP
antibody for 1 hour at room temperature. Results show that biotin was only detected in the RNA pol II phospho Ser2 non-cleavable biotin antibody (Lane 1) and the RNA pol II phosphor Ser5 non-cleavablebiotin antibody (Lane 3). The antibodies labeled with the HRV 3C biotin and digested with the protease (Lanes 2 and 4) no longer had biotin attached.

Troubleshooting
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 may prohibit theaddition of 2 labels per antibody molecule depending on the label used). Commercially available biotin quantification kits are designed to detect
antibodies that have been chemically labeled and, therefore, contain a higher number of biotin molecules per antibody. Attempting to detect
only 2 biotins per antibody will fall below the limit of detection and we do not suggest using these biotin quantification kits. Instead, we suggest afunctional assay to detect the presence of the biotin labeled antibody. (e.g. Western blot with Streptavidin detection)

Can I label for shorter orlonger time periods?

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

Can I label smaller
amounts of 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-Glycinelabel should not be modified in the reaction. Volume differences should be corrected using Reaction Buffer AM3 to maintain a total volume of 200 µl
per reaction. Optimization of labeling time may be required. Please note that the yield of recovery 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 the excess label and Sortase A5 prior to use. If the antibody will be used immediately, skipping purification may be acceptable. However, storage of unpurified antibody over time will result in removal of the label. Any active
Sortase A5 will continue to cleave at the recognition sequence and may create large antibody complexes or completely remove the poly-Glycinelabels resulting in unlabeled antibody. Therefore, purification prior to use and storage is strongly recommended.

North America 877 222 9543

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

www.activemotif.com

Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of the numbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008
USAToll Free: 877 222 9543
Telephone: 760 431 1263
Fax:
760 431 1351
E-mail:tech_service@activemotif.com
Active Motif Europe
Avenue Reine Astrid, 92
B-1330 La Hulpe, Belgium
UK Free Phone:
0800 169 31 47
France Free Phone: 0800 90 99 79
Germany Free Phone:
0800 181 99 10
Telephone:+32 (0)2 653 0001
Fax:
+32 (0)2 653 0050
E-mail:eurotech@activemotif.com
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone: +81 3 5225 3638
Fax:
+81 3 5261 8733
E-mail:japantech@activemotif.com
Active Motif China
787 Kangqiao Road
Building 10, Suite 202, Pudong District
Shanghai, 201315, China
Telephone: (86)-21-20926090
Hotline:400-018-8123
E-mail:techchina@activemotif.com
Visit Active Motif on the worldwide web at http://www.activemotif.com
At this site:• Read about who we are, where we are, and what we do• Review data supporting our products and the latest updates• Enter your name into our mailing list to receive our catalog, MotifVations newsletter and notification of our upcoming products• Share your ideas and results with us• View our job opportunities
Don’t forget to bookmark our site for easy reference!

North America 877 222 9543

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

www.activemotif.com