Overview

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Upon receipt, store the kit at 4 ^oC. When stored properly, the kit should be stable for six months. Alternatively Components A and B can be stored at -20°C. Do not freeze 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 goat anti-mouse IgG antibody.

1. Prepare antibody solution :

For labeling 100 µg antibody (assuming the target antibodyconcentration is 1 mg/mL), mix 5 µL (5% of the total reaction volume) of Reaction Buffer (Component C) with 100 µL of the target antibody solution.

Note 1. If you have a difference  concentration, adjust the antibody volume accordingly to make ~100 µg antibody available for your labeling reaction.

Note 2: The antibody should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4; If the antibody is 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 (cat# UFC501008 from Millipore) to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for antibody precipitation.

Note 3: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well.

Note 4: The antibody –Buccutite™ MTA reaction efficiency is significantly reduced if the antibody concentration is less than 1 mg/mL. For optimal labeling efficiency the final antibody concentration range of 1-10 mg/mL is recommended.

2. Run Antibody-Buccutite™ MTA reaction:

2.1    Add the antibody solution directly into the vial of Buccutite ™ MTA (Component B), and mix them well by repeatedly pipetting for a few times or vortex the vial for a few seconds.

2.2    Keep the antibody- Buccutite ™ MTA reaction mixture at room temperature for 30 - 60 minutes.

Note: The antibody-Buccutite™ MTA reaction mixture can be rotated or shaken for longer time if desired.

3. Prepare spin column for antibody-Buccutite™ MTA purification:

3.1    Invert the provided spin column (Component D) several times to re-suspend the settled gel and remove any bubbles.

3.2    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).

3.3    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.

3.4    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.

3.5    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.

4. Purify the antibody-Buccutite™ MTA solution:

4.1    Place the column (from Step 3.5) in a clean Collecting Tube (1.5 mL, not provided). Carefully load the sample (~105 μL, from Step 2.2) directly to the center of the column.

4.2    After loading the sample, add 5 μL of 1X PBS (pH 7.2-7.4) to make the total volume of 110 μL. Centrifuge the column for 5-6 minutes at 1,000 x g, and collect the solution that contains the desired antibody-Buccutite™ MTA solution.

5. Make HRP-antibody conjugation:

5.1    Make HRP- Buccutite™ FOL solution by adding 50 μL ddH₂O into the vial of HRP- Buccutite™ FOL (Component A), mix well by repeatedly pipetting for a few times or vortex the vial for a few seconds.

5.2    Mix whole vial of HRP- Buccutite™ FOL solution (from Step 5.1) into the purified antibody- Buccutite™ MTA solution (from Step 4.2), mix well and rotating the mixture for 1 hour at room temperature.

5.3    The HRP-antibody conjugate is now ready to use.

Note 1: For immediate use, the HRP-antibody conjugate need be diluted with the buffer of your choice.

Note 2: For longer term storage, HRP-antibody conjugate solution need be concentrated or freeze dried.

References & Citations

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