TrailBlazer Tag Kit

12020038

1. Antibody preparation
   1. Add 100 µg of antibody to the filter and place into the collection tube.
   2. Add the amount of Conjugation Buffer required to reach 500 µl total volume. Cap and invert to mix.
   3. Centrifuge at 5000 x g until the volume reaches ~50 µl (approximately 5 min). Discard the eluate in the collection tube.
   4. Repeat steps 1.2 - 1.3 two additional times.
   5. Ensure that the volume of the antibody is ~50 µl and no more than 100 µl. If required perform further centrifugations. Invert the filter and place into the empty collection tube.
   6. Centrifuge the tube with the inverted filter for 30 sec at 5000 x g. Retain the eluate that contains the antibody.
2. Antibody modification
   1. Prepare Reagent A by adding 75 µl of Conjugation Buffer.
   2. Add 2 µl of prepared Reagent A to the antibody solution, briefly vortex to mix.
   3. Incubate at room temperature for 60 min.
   4. During the incubation prepare a desalting column.
      1. Snap open the bottom of the column and insert the column into a 2 ml collection tube. Remove and discard the column cap and centrifuge for 2 min at 1000 x g. Discard eluate.
      2. Apply 500 µl of Conjugation Buffer and centrifuge for 1 min at 1000 x g. Discard eluate.
      3. Repeat step 2.4.2 for two additional times. Add Conjugation Buffer for the third wash but wait to centrifuge until immediately prior to proceeding to step 2.5. After the third wash check there is no solution in the column, if required briefly centrifuge until no solution remains.
   5. Insert prepared desalting column into a fresh 1.5 ml microcentrifuge tube and apply the entire sample from step 2.3 to the column.
   6. Centrifuge at 1000 x g for 4 min and retain eluate.
3. Attachment of SpyTag to the antibody
   1. Prepare Tag by adding 5 µl of ultrapure water and vortex briefly.
   2. Add 2 µl of prepared Tag to the prepared antibody, vortex briefly and pulse spin.
   3. Incubate at room temperature for 30 min – 3 hours.
   4. During the incubation prepare a second desalting column, following steps 2.4.1 – 2.4.3.
   5. Insert prepared desalting column into a fresh 1.5 ml microcentrifuge tube and add the entire sample from step 3.3 to the column.
   6. Centrifuge at 1000 x g for 4 min and retain eluate that contains the modified antibody.

This completes the TrailBlazer Tag Kit Protocol. Proceed to using the TrailBlazer Label Kit or store the SpyTagged antibody at +4°C for up to 2 weeks.

For full instructions for use please go to: bio-rad-antibodies.com/tag-kit-IFU.