Our on-column antibody labeling protocol relies on the very short diffusion distances within the pores of our Capturem membranes. These short distances allow for extremely rapid conjugation reactions when antibodies are loaded onto a Capturem Protein G Miniprep Column and labeling reagents are then centrifuged through the column in an appropriate buffer.

• Capturem Protein G Miniprep Columns (Cat. # 635725)
• Protein G binding buffer:
  • To obtain optimal and reproducible performance, we recommend using Pierce Protein G IgG Binding Buffer (Cat. # 21019).
  • You can also use 20–100 mM sodium acetate containing 0.15–2 M NaCl, pH 5.0, or 100–500 mM sodium phosphate containing 0.15–2 M NaCl, pH 7.0.
• NHS-Fluorescein (Thermo Fisher, Cat. No. 46410)
• Neutralization buffer (1M Tris, pH 8.5)
• 1x PBS (pH 7.4)
• Elution buffer (0.1 M glycine, pH 2.5)

Your antibody sample should be in an appropriate binding buffer before it is loaded onto the Capturem Protein G Miniprep Column. If not, dilute a sample containing 100 µg of your antibody with one of the recommended Protein G binding buffers mentioned above. The dilution should be at least 1:2 (initial:final volume). If this results in a volume greater than 1 ml, you can split your sample and load the antibody onto the column in multiple steps.

1. Equilibrate a Capturem Protein G Miniprep Column by adding 300 µl of binding buffer to the column and centrifuging at 1,000g for 1 min at room temperature. Discard the flowthrough.
2. Load 100–800 µl of your antibody solution onto the equilibrated column and centrifuge at 1,000g for 1 min at room temperature. Save the collection tube containing the sample flowthrough for antibody analysis and transfer the column to a new collection tube.

This protocol describes an NHS-fluorescein labeling reaction, which is performed in PBS. It can easily be adapted to other NHS-based chemistries, including NHS-biotin. For other bioconjugation chemistries, replace the PBS used in the initial wash step and subsequent steps with a buffer compatible with your reagent. If you are interested in labeling thiols, e.g., with a maleimide reaction, you can add a TCEP reduction step after the initial wash.

1. Add 200 µl of PBS to the Capturem Protein G Miniprep Column and centrifuge at 1,000g for 1 min at room temperature to wash the column.
2. Dissolve NHS-Fluorescein in DMSO at a concentration of 10 mg/ml. Dilute 20 µl of this stock into 800 µl of PBS immediately before use, then load all of the NHS-Fluorescein solution onto the column and spin at 500g for 3 min.
3. Wash away excess NHS-Fluorescein by loading 400 µl of PBS onto the column and centrifuging at 1,000g for 1 min at room temperature. Repeat this step once.

1. Add 10 µl of neutralization buffer to a clean collection tube. Elute your antibody by loading 45 µl of elution buffer onto the Capturem Protein G Miniprep Column and centrifuging at 1,000g for 1 min. Perform a second elution by repeating this step, then combine and thoroughly mix the fractions.
2. Your antibody is now ready for analysis and use in downstream applications.

For more information, refer to the Capturem Protein G Miniprep Protocol-At-A-Glance.