Experiment #1 - Antibody Purification

Covalent conjugation using EDC and sulfo-NHS require proteins to be suspended in an amine-free buffer that will not interfere with conjugation to the primary amines in the protein lysine residues. Commercial antibodies often contain salts and protein additives for stabilization (e.g. BSA), amines as a preservative (e.g. sodium azide), or amines in the buffer (e.g. tris) that require removal before conjugation. Even small concentrations of sodium azide can interfere with the efficacy of conjugation and many antibody solutions contain residual tris from the isolation/purification process that is not noted on the certificate of analysis.
Scientist Extracting a Rack Tube With Urine Samples. Closeup of a Scientist Working With Urine Samples in Lab.
It is important to distinguish the antibody purification referred to in this protocol from the affinity purification during antibody development and processing. Many antibodies are supplied purified via protein A, protein G, affinity purification columns, or other methods. Tris buffer is commonly used to elute antibodies from purification columns and these "affinity purified" antibodies still need to be purified away from free amines. Unless you are confident that the antibody is in an amine-free buffer, we recommend that all antibodies be purified before conjugation.

Fortis Life Science recommends the use of a centrifugal purification method for most IgG antibodies and other proteins. We recommend selecting a purification column with a molecular weight cut-off below that of the antibody but above any stabilizing proteins that may be present in the antibody solution. The antibody or protein should be concentrated, washed, collected, and resuspended to a concentration of ≥ 1 mg/mL in an amine-free buffer.

Technical Notes

The first step in antibody purification is to select an antibody purification buffer. In our optimization kit we include a 10 mM potassium phosphate buffer at pH 7.4. This is a common buffer that antibodies are supplied in by many vendors. If your antibody is supplied in a buffer that has a significantly different pH than 7.4, an alternative protein buffer may produce better results and we recommend contacting the antibody vendor to select an appropriate antibody purification buffer.

We also recommend the following points:

  • Purify at least 0.5 mg of material for use in the subsequent conjugations to Fortis Life Science BioReady™ 150 nm Carboxyl Gold Nanoshells.
  • Perform all steps as quickly as possible (< 90 minutes). Leaving concentrated protein sitting in the purification filter for extended periods may result in low yields. If possible perform purification in a refrigerated centrifuge at 4°C.
  • A very small volume of solution should remain in the filter after each spin (~30–50 µL). This contains the antibody; spinning until all the solution has gone through may cause adsorptive loss of antibody on the membrane.

Materials

We recommend using the following - or similar - materials:

  • Antibody purification filter (Millipore Amicon® Ultra 0.5 mL Filters 10kDa, Cat# UFC501096)
  • 2 × 2 mL microcentrifuge tubes
  • Antibody purification buffer - 10 mM potassium phosphate, pH 7.4

Antibody Purification Flow Chart

Figure 1 shows the methodology overview for the 13 steps required during the antibody purification experiment, with further details from the protocol outlined below:

Antibody Purification Protocol

  1. Confirm that the antibody concentration matches that provided on the antibody Certificate of Analysis using A280, BCA, or a Bradford protein assay.
  2. Place the filter device inside of one of the provided 2 mL microcentrifuge tubes.
  3. Pre-wet the filter device by adding 450 µL of the appropriate purification buffer to the filter and centrifuge for 5 minutes at 13.8k RCF.

    Note: Do not touch the inside of the filter with the pipette as this may risk puncturing the filter.

  4. Carefully remove the filter device from the microcentrifuge tube and dispose of filtrate located at the bottom of the microcentrifuge tube. Place the filter device back into the microcentrifuge tube after the filtrate has been removed.
  5. Add antibody solution into the filter and close cap.

    Note: The filter can hold up to 500 µL. If the volume to purify exceeds this capacity, you can centrifuge 450 uL of antibody solution in the filter first to concentrate and then add additional unpurified antibody before continuing with the wash steps. If the antibody volume is minimal before any centrifugation step, make up the volume by adding enough purification buffer to bring the total volume up to ~450 µL.

  6. Centrifuge 5 minutes at 8k RCF to concentrate.
  7. Remove the tube from the centrifuge and remove the filter device from the tube. Remove filtrate.

    Note: It is recommended to retain the antibody filtrate until after you have determined the yield. In the event of a ruptured filter, you may be able to recover your antibody from the filtrate.

  8. Place the filter containing the concentrated antibody back into the tube and add 350 µL of purification buffer to the filter.
  9. Centrifuge for 5 minutes at 8k RCF to wash and concentrate. Remove filtrate after spin.
  10. Repeat washing procedure (Steps 8–9) an additional four times using 350 µL of additional purification buffer for a total of five
  11. Following the final wash, turn the filter device upside down in a new, clean 2 mL microcentrifuge tube (provided).
  12. Centrifuge 5 minutes at 0k RCF to collect purified and concentrated antibody.

    Note: The microcentrifuge tube cap may be cut off with scissors before spinning.

  13. Bring the collected antibody solution to a final volume such that your antibody concentration is ≥ 1 mg/mL for storage (recommended).

    Note: After purification, it may be desirable to aliquot and store purified antibodies ≥ 1 mg/mL at recommended storage temperature to minimize risk of contamination and to minimize freeze/thaw cycles. Antibody and protein stability conditions vary. Always refer to the protein data sheet provided by the supplier for proper storage and handling.
antibody purification experimentation flow chart
Figure 1 - antibody purification experimentation flow chart

Determine Final Protein Concentration

Use A280 method, BCA, or Bradford assay to confirm the concentration of starting material and final purified material. With these values you can determine yield. It is recommended to use the same metric of quantification that was provided with the protein data sheet from the supplier.

Your protein is now ready for use in covalent conjugation and subsequent optimization. Store antibody at 2–8°C prior to conjugation.

Next Steps

Proceed to experiment #2 - Reaction Buffer Screen