HRP Antibody Labeling Kit

The HRP antibody labeling kits have been developed to modify 90-110 ug of purified antibody at 0.9-1.1 mg/ml, incorporating the optimal range of HRP. Easy to use with efficient labeling, generating conjugates which may be used in ELISA, western blotting or 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 extraneous proteins such as BSA, serum or gelatin. Antibody formulations containing tris, 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 the provided collection tube, then place in a 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 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 the provided collection tube, 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 a new collection tube and centrifuge for 2 minutes at 1500 g. Labeling with HRP 7) Add flow-through from GREEN-capped column collection tube to HRP 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 HRP 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 the provided collection tube, then place in a 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 the final buffer tube (Part G) and centrifuge for 2 minutes at 1500 g. 11) You now have an HRP conjugated antibody ready to use. The final concentration of antibody will be ~0.6-0.7 mg/mL based on volume and approximated antibody recovery. Please store final conjugate at 2-8 0C.