Buccutite™ Bioconjugation Technology

Introduction

---

SMCC heterobifunctional crosslinkers are often used in the preparation of PE, APC and tandem dye antibody conjugates. Although and established technique, SMCC-based conjugation suffers from several drawbacks, including: (1) poor conjugation efficiency (~30% yield), (2) requires antibody reduction using DTT, which may significantly impact antibody immunoreactivity, and (3) requires very high concentrations of reactants (i.e. PE and antibody) for adequate reactivity.

An alternative to SMCC conjugation is AAT Bioquest's Buccutite™ technology. Buccutite™ phycobiliprotein labeling kits (Table 2) provide a fast, simple and efficient technique for labeling PE, APC or tandem dyes to antibodies in microscale. It utilizes two separate linkers, Buccutite™ MTA and Buccutite™ FOL. These linkers are independently labeled to the phycobiliprotein and antibody of interest, and when mixed will bind strongly together resulting in an antibody-phycobiliprotein conjugate.

The following is a general guideline for labeling 100 µg of antibodies using AAT Bioquest's Buccutite™ Rapid Phycobiliprotein Antibody Labeling Kits *Microscale Optimized for Labeling 100 µg Antibody per Reaction*. This labeling protocol is not suitable for antibodies containing bovine albumin serum (BSA), gelatin, free amino acids or ammonium salts. Antibodies or proteins containing any of these impurities will have a very low conjugate yield, and as such require extensive purification.

Buccutite™ Kit Components

---

1. Upon receipt, store the kit at 4°C. When stored properly, the kit should be stable for six months. Alternatively, Component B can be stored at -20°C. Do not freeze Buccutite™ FOL-Activated PE (Component A), Reaction Buffer (Component C) and Spin Column (Component D). Warm all the components and centrifuge the vials briefly before opening, and immediately prepare the required solutions before starting your conjugation. The following SOP is an example for labeling IgG antibody.

Materials Required (Not Supplied)

---

• 100 µg unconjugated antibody
• 1X PBS buffer (pH 7.2 – 7.4) [for recipe, click here]
• 2 mL washing tube
• 5 mL collecting tube
• 12 x 75 mm test tube (optional)
• Centrifuge
• NanoDrop^® Spectrophotometer

Preparation

---

Antibody Working Solution Preparation

1. In a suitable container, dissolve 100 µg of your antibody with 100 µL of 1X PBS buffer (pH 7.2 – 7.4) to make a 1 mg/mL antibody solution.
   *Note: If you have a different concentration, adjust the antibody volume accordingly to make ~100 µg antibody available for your labeling reaction.
2. Add 5 µL (5% of the total reaction volume) of Reaction Buffer (Component C) to your antibody solution.
   *Note: If your antibody was dissolved in glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, or use Amicon Ultra-0.5, Ultracel-10 Membrane, 10 kDa (Millipore Cat No. UFC501008) to remove free amines or ammonium salts (i.e. ammonium sulfate and ammonium acetate) that are widely used for antibody precipitation.

Buccutite™ MTA – Antibody Preparation

1. Add the antibody solution directly into the vial of Buccutite™ MTA (Component B) and mix or vortex thoroughly.
2. Continuously rotate or shake reaction mixture at room temperature (37°C) for 60 minutes.

Purification

---

Prepare spin column for antibody-Buccutite™ MTA purification

1. Invert the provided spin column (Component D) several times to re-suspend the settled gel and remove any bubbles.
2. Snap off the tip and place the column in a washing tube (2 mL). Remove the cap to allow the excess packing buffer to drain by gravity to the top of the gel bed. If column does not begin to flow, push cap back into column and remove it again to start the flow. Discard the drained buffer, and then place the column back into the Washing Tube. However, centrifuge immediately if the column is placed into a 12 x 75 mm test tube.
3. Centrifuge for 2 minutes in a swinging bucket centrifuge at 1,000 x g (see Centrifugation Notes section) to remove the packing buffer. Discard the buffer.
4. Apply 1-2 mL 1X PBS (pH 7.2-7.4) to the column. After each application of PBS, let the buffer drain out by gravity, or centrifuge the column for 2 minutes to remove the buffer. Discard the buffer from the collection tube. Repeat this process 3-4 times.
5. Again, centrifuge for 2 minutes in a swinging bucket centrifuge at 1,000 x g to remove the packing buffer. Discard the buffer.

Purify the antibody – Buccutite™ MTA solution

1. Place the spin column in a clean 1.5 mL collecting tube.
2. Carefully load the antibody – Buccutite™ MTA solution (~105 µL) directly to the center of the column.
3. After loading sample, add 5 µL of 1X PBS (pH 7.2 – 7 .4) to the center of the column.
   *Note: Total volume should equal ~110 µL
4. Centrifuge column for 5 – 6 minutes at 1,000 x g.

Conjugation Procedure

---

Run antibody – phycobiliprotein conjugation reaction

1. Add 50 µL of ddH₂0 into the vial containing Buccutite™ FOL – Activated phycobiliprotein (Component A), and mix or vortex thoroughly.
2. Add the entire vial of Buccutite™ FOL – Activated phycobiliprotein solution to the purified antibody – Buccutite™ MTA solution, and mix or vortex thoroughly.
3. Continuously rotate or shake conjugation reaction mixture for 1 hour at room temperature (37°C).
4. Antibody-phycobiliprotein conjugate is now ready to use.
   *Note: For immediate use, first dilute the antibody-phycobiliprotein conjugate in a buffer of your choice.

Storage Conditions

• The antibody-phycobiliprotein conjugates should be stored at > 0.5 mg/mL in the presence of a carrier antibody (e.g. 0.1% bovine serum albumin).
• The antibody-phycobiliprotein conjugate solution can be stored at 4°C for two months when stored in the presence of 2 mM sodium azide and protected from light.
• For long-term storage, the antibody-phycobiliprotein conjugates should be lyophilized and stored at ≤ –20°C.

Additional Resources

---

Centrifugation Notes

Spin column (Component D) can fit into 2 mL micro-centrifuge tubes or 12 x 75 mm test tubes for sample collection during centrifugation. Use the 2 mL micro-tube with the columns for the initial column equilibration step.

Swinging bucket centrifuges capable of generating a minimum force of 1,000 x g are suitable for Bio-Spin column use. The gravitational force created at a particular revolution speed is a function of the radius of the micro-centrifuge rotor. Consult the swinging bucket centrifuge instruction manual for the information about conversion from revolutions per minute (RPM) to centrifugal or g-force. Alternatively, use the following equation to calculate the speed in RPM required to reach the gravitational force of 1,000 x g.

RCF (x g) = (1.12 x 10-5) × (RPM)×2×r (RCF is the relative centrifugal force, r is the radius in centimeters measured from the center of the rotor to the middle of the Bio-Spin column, and RPM is the speed of the rotor).