Buccutite™ Rapid Protein Crosslinking Kit *Microscale Optimized for Crosslinking 100 ug Antibody Per Reaction*

1. Prepare protein solutions
2. Run protein-1 Buccutite™ MTA reaction
3. Run protein-2 Buccutite™ FOL reaction
4. Cross-link protein-1 and protein-2 reactions by mixing and incubating at room temperature

Upon receipt, store components A and B at -20 °C. When stored properly, the kit should be stable for six months. Do not freeze the Reaction Buffer (Component C) and Spin Column (Component D). Warm all the components and centrifuge the vials briefly before opening them. Immediately prepare the necessary solutions before starting your conjugation. The following SOP is an example for conjugating 100 µg protein-1 with protein 2.

Note: Key parameters to achieve the best performance:

1. Protein molecular weight: >100,000 Daltons
2. Protein concentration: >= 1mg/mL
3. Protein sample volume: 60~120 µL

For labeling 100 µg protein-1 and protein-2 (assuming the concentrations are 1 mg/mL for both proteins), mix 5 µL (5% of the total reaction volume) of Reaction Buffer (Component C) with 100 µL of each protein solution.

Note: The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4. If it is dissolved in glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, or use ReadiUse™ 10KD Spin Filter (Cat No. 60502 from AAT Bioquest) to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation.

Note: Stabilizers like bovine serum albumin (BSA) or gelatin will affect the labeling reaction.

1. Add 105 µL Protein-1 solution directly into the vial of Buccutite ™ MTA (Component A), and mix them well by repeatedly pipetting for a few times or vortex the vial for a few seconds.
2. Keep the Protein-1- Buccutite ™ MTA reaction mixture at room temperature for 30 - 60 minutes. The Protein-Buccutite™ MTA reaction mixture can be rotated or shaken for longer time if desired.
3. Purify Protein-1- Buccutite ™ MTA through desalting column. Please refer to “Prepare spin column for Protein purification” part for detailed operations.
4. Calculate the concentration of the Protein-1- Buccutite ™ MTA with 75% yield after desalting. (For example: if starting with 100 µg protein, after desalting column purification, the recovery protein amount is ~75 µg.)

1. Add the 105 µL Protein-2 solution directly into the vial of Buccutite ™ FOL (Component B), and mix them well by repeatedly pipetting for a few times or vortex the vial for a few seconds.
2. Keep the Protein-2- Buccutite ™ FOL reaction mixture at room temperature for 30 - 60 minutes. The Protein-Buccutite™ FOL reaction mixture can be rotated or shaken for longer time if desired.
3. Purify Protein-2- Buccutite ™ FOL through desalting column. Please refer to “Prepare spin column for Protein purification” part for detailed operations.
4. Calculate the concentration of the Protein-2- Buccutite ™ FOL with 75% yield after desalting.

1. Cross-linking reaction is initiated by mixing Protein-1- Buccutite ™ MTA and Protein-2- Buccutite ™ FOL at the desired molar ratio. Usually, Buccutite ™ FOL-modified Protein-2 is mixed with Buccutite ™ MTA-modified Protein-1 in 1.5-2.0 molar ratio to drive the crosslinking reaction to completion. The mixing ratio can be reversed depending on your downstream applications, and the cost of your proteins.
2. Rotate the mixture for 1~2 hour at room temperature or stay at 4 °C overnight. The reaction mixture is now ready to use, or to be stored at 4 °C. Desalting is optional.

1. A small amount of reaction mixture (for example: 2~4 mg) could be analyzed using 4-12% Bis-Tis Protein Gel in a SDS running buffer system to check the conjugation result.
2. The conjugation reaction mixture contains the desired conjugate along with small amount of unlinked Protein-2 (used in excess). If required, the reaction mixture can be purified by size exclusion chromatography (SEC), and the desired conjugate fractions are pooled and combined.

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, not provided). 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 (not provided).
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 for 3-4 times.
5. 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.
6. Place the column in a clean Collecting Tube (1.5 mL, not provided). Carefully load the sample (~105 µL) directly to the center of the column.
7. After loading the sample, add 10 µL of 1X PBS (pH 7.2-7.4), centrifuge the column for 5-6 minutes at 1,000 x g, and collect the solution that contains the desired protein-1-Buccutite™ MTA solution or Protein-2- Buccutite ™ FOL solution.

Spin column (Component D) can fit into 2 mL microcentrifuge tubes or 12 x 75 mm test tubes for sample collection during centrifugation. Use the 2 mL microtube 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 microcentrifuge 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

RPM is the speed of the rotor