Sortag-IT™ ATTO 647 Labeling Kit
Catalog No.: 13110
Format:  3 x 100 µg
Description
The Sortag-IT™ ATTO 647 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.

Contents
Each Sortag-IT™ ATTO 647 Labeling Kit provides sufficient materials to label 3 x 100 µg AbFlex® recombinant antibody with a yield > 50%.
• 3 units Sortase A5 enzyme (1 unit/µl); Store at -80°C• 1 ml Reaction Buffer AM3; Store at 4°C• 1.53 mM (Gly)5- ATTO 647 label (1.53 mM = 1.95 mg/ml); Store at -20°C• 10 µl Stop Solution AM3; Store at RT• 3 ea Purification columns; Store at RT
Items 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 ATTO 647:
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. Keep tube wrapped in foil and protected by light during the labelingreaction. Add reagents in the order shown below and mix by pipetting.
Reagent

Volume to add

AbFlex antibody (1 µg/µl)

100 µlReaction Buffer AM383 µl(Gly)5-ATTO label (1.53 mM)

16 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. Keep sample protected from light during incubation.
4. Following the incubation, remove excess label from the reaction by purification using the included columns. Keep samples protectedfrom light as much as possible.
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. Wrap the new collection tube in foil and protect from light.
5. Add 2.5 µl Stop Solution AM3 to each labeled antibody to inactivate the Sortase A5 enzyme.
6. Use immediately, or store in the dark at 4°C for up to 3 months.

North America 877 222 9543

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

www.activemotif.com

Appendix
Application Data

Immunofluorescence staining using ATTO 647 directly conjugated antibody.
The Sortag-IT™ ATTO 647 Labeling Kit was used to directly conjugate 100 µg of Active Motif’s AbFlex® Lamin A/C recombinant antibody with ATTO 647. Staining shows nuclear localization of the Lamin A/Cantibody as expected.

Troubleshooting
Problem/question

Recommendation

What is the excitation
range for ATTO 647?

ATTO 647 fluorescence is excited most efficiently in the range of 625 - 660 nm.

How can I determine thelabeling efficiency?

For ATTO 647 the Degree of Labeling (DOL) can be determined using Lambert-Beer law.
The maximum number of labels that can be added to an AbFlex antibody is 2 (this is atheoretical maximum as steric hindrance may prohibit the addition of 2 labels per antibody molecule depending on the label used). We anticipate a DOL value greater than 1.0
DOL = Amax / Emax                =                     Amax X  Eprot           Aprot / Eprot                               (A280 - Amax X CF) X Emax
A280 = absorbance of conjugate solution measured at 280 nm.
Amax = absorbance of conjugate solution measured at lEx (646 nm for ATTO 647)
CF = correction factor = A280/Amax (0.03 for ATTO 647)
Eprot = extinction coefficeint for antibody (210,000 M-1cm-1 for IgG)
Emax = extinction coefficeint for ATTO dye (150,000 M-1cm-1 for ATTO 647)

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-Glycinelabel 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-Glycine label 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 berequired. Please note that the yield of recovery for smaller antibody amounts may bediminished.

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 inremoval 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 isstrongly 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.
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North America 877 222 9543

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

www.activemotif.com