R-PE Antibody Labeling Kit

The R-PE antibody labeling kits have been developed to modify 90-110 μg of purified antibody at 0.9-1.1 mg/ml. The kits are easy to use and deliver a high labeling efficiency, generating R-PE conjugates which may be used in flow cytometry, immunofluorescence and immunohistochemistry. 1. All components are included in each kit 2. The only equipment needed are a pipette and a microcentrifuge 3. Please ensure your antibodies for labeling are at 0.9-1.1 mg/ml in buffer free of Tris and extraneous proteins such as BSA, serum or gelatin. Antibody formulations containing azide, trehalose or other sugars are acceptable.

Antibody Preparation 1) Antibody preparation- Prepare a solution of 90-110 μg antibody at 0.9-1.1 mg/mL- Nanodrop absorbance 0.126-0.150 au (1.0 cm-1 path) or plate reader. 2) Desalting column preparation- Break off bottom of BLUE-capped column and untighten the BLUE cap and place column (s) in collection tubes, then place in microcentrifuge and spin for 2 minutes at 1500 g. Discard flow-through and collection tube. 3) Add antibody directly to the BLUE-CAPPED column, replace BLUE cap lightly on column, insert in a new collection tube and centrifuge for 2 minutes at 1500 g. 4) Add flow-through from BLUE-capped column to Antibody Linker Reagent (Part D), pipette several times to mix, lightly vortex and incubate for 1 hour at room temperature. Antibody Desalting 5) Desalting column preparation- Break off bottom of GREEN-capped column and untighten the GREEN cap and place column (s) in collection tubes, then place in microcentrifuge and spin for 2 minutes at 1500 g. Discard flow-through and collection tube. 6) Add linker-modified antibody solution to GREEN-capped column, replace GREEN cap lightly on column, insert in a new collection tube and centrifuge for 2 minutes at 1500 g. Labeling with R-PE 7) Add flow-through from GREEN-capped column collection tube to the R-PE labeling reagent tube (Part E), pipette several times to mix and lightly vortex. 8) Take 15 μL of Link Buffer (Part F) and add directly to the R-PE labeling reagent tube which already contains your antibody, pipette several times to mix, lightly vortex, and incubate for 1 hour at room temperature in the dark. Purification 9) PBS desalting column preparation - Break off bottom of RED-capped column and untighten the RED cap, place column in collection tube, then place in microcentrifuge and spin for 2 minutes at 1500 g. Discard flow-through and collection tube. 10) Transfer labeling mixture to the RED-capped tube from Step 7, insert in a new collection tube and centrifuge for 2 minutes at 1500 g. 11) You now have a R-PE conjugated antibody ready to use. The final concentration of the antibody will be ~0.6-0.7 mg/mL. Please store final conjugate at 2-8 0C.