Amplite® Rapid Colorimetric Maleimide Quantitation Kit *Optimized for Quantifying APC Maleimide*

Upon receipt, store the vials of Maleimide 555™ (Component A) at -20 °C (preferred at -80°C), kept from light and moisture. When stored properly, the kit components should be stable for six months. 

Note: Do not freeze the Spin Columns (Component C). 

Note: Warm all the components before running the required assays. 100 μg APC-maleimide is needed to determine the molar ratio of maleimide to APC.

1. Obtain a 100 µg sample of APC-maleimide.

2. Adjust the volume to 100 μL using Assay Buffer (Component B). 

   Note: Make sure the APC-maleimide sample is prepared in a pH 6.0 buffer and without any free maleimide. 

1. From the 'Prepare APC-Maleimide Sample Solution' section, take the solution prepared in Step 2 and add it to a vial of Maleimide 555™ (Component A). Mix well by repeatedly pipetting a few times or vortexing the vial for a few seconds.

2. Keep the reaction mixture at room temperature and rotate for 30 - 60 minutes.

1. Invert the Spin Column (Component C) several times to suspend the settled gel and remove any bubbles.

2. Snap off the tip and place the column in a 2 mL Washing Tube (not provided). Remove the cap to let the excess packing buffer drain by gravity until it reaches the top of the gel bed. If the column doesn't start to flow, reinsert and remove the cap to initiate drainage. Dispose of the buffer. Place the column back into the Washing Tube. However, if the column is placed into a 12 x 75 mm test tube (not provided), centrifuge immediately.

3. Centrifuge for 1 minute 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 mL of the Assay Buffer (Component B) to the column, let the buffer drain out by gravity, or centrifuge the column for 1 minute 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 reaction buffer. Discard the buffer.

1. Insert the column from Step 5 of the 'Prepare the Spin Column for Sample Purification' section into a clean 1.5 mL Collecting Tube. Gently pipette the sample (100 μL) directly into the center of the column.

2. After loading the sample, add 10 μL of Assay Buffer (Component B) to the top of the column. Then centrifuge the column for 5 minutes at 1,000 x g, and collect the solution in the collecting tube.

1. Dilute the maleimide reaction product obtained in Step 2 from the 'Purify Maleimide Reaction Product' section. Use 5 to 10 times the volume of Assay Buffer (Component B) for this dilution. The exact amount of buffer required will depend on the size of the cuvette and the absorbance measurement obtained.

   Note: The dilution factor doesn’t affect the final maleimide quantitation result.

   Note: Dilution is not needed if using Nanodrop to measure Absorbance

2. Measure the absorption spectrum from 900 nm to 250 nm range, or only read the absorbance number at 650 nm and 555 nm. 

Calculations:

1. Calculate the change in absorbance

ΔA = (A₅₅₅ - A₆₅₀)

Where:

• A₅₅₅ = Absorbance of APC-maleimide sample at 555 nm
• A₆₅₀ = Absorbance of APC-maleimide sample at 650 nm

2. Calculate the amount of APC-maleimide

Moles of Maleimide/Moles of protein or antibody = [ΔA x CF₅₅₅) ÷ (ε_{Maleimide 555™})] ÷ [(A₆₅₀) ÷ (ε_APC)]

Where:

• ε_{Maleimide 555™} = 150,000 M^-1cm^-1
• ε_APC = 730,000 M^-1cm^-1
• CF₅₅₅ = 0.157
• A₆₅₀ = Absorbance of APC-maleimide sample at 650 nm

Spin Column (Component C) can fit into 2 mL microcentrifuge tubes or 12 x 75 mm test tubes for sample collection during centrifugation. Use the 2 mL microtubes provided 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 information about conversion from revolutions per minute (RPM) to centrifugal or g-force. Alternatively, the speed in RPM required to reach the gravitational force of 1,000 x g can be calculated using the following equation:

RCF (g) = (1.2 x 10^-5 x (RPM)² x r

• RCF = the relative centrifugal force
• RPM = the speed of the rotor
• r = the radius in centimeters measured from the center of the rotor to the middle of the Bio-Spin column