TITLE: Maxpar MCP9 cadmium labeling
AUTHORS: Albert Tsai

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Antibody labeling with cadmium isotopes</div></div>

[STEPS]
?. [Preparation]
Bring all buffer solutions to room temperature.
?. [Polymer loading and wash 1]
Thaw Maxpar MCP9 polymer to room temperature before opening to avoid moisture condensation.
?. [Preparation]
Centrifuge the stock antibody at 12,000 × g for 5 minutes to sediment antibody aggregates, and then verify the stock antibody concentration by spectrophotometry.
?. [Preparation]
Ensure that the water bath and dry heat block are equilibrated to 37 °C.
?. [Polymer loading and wash 1]
Centrifuge the MCP9 polymer tube and the tube containing 50 mM cadmium nitrate for 10 seconds in a minicentrifuge.
?. [Polymer loading and wash 1]
Add 87 μL of L buffer to the MCP9 polymer tube to resuspend the polymer. Mix thoroughly by pipetting until the polymer is completely dissolved (approximately 1 min).
?. [Polymer loading and wash 1]
Add 13 μL of 50 mM cadmium metal solution to the MCP9 polymer tube. Mix thoroughly by pipetting.
?. [Polymer loading and wash 1]
Incubate at 37° C for 60 minutes in a water bath or dry heat block. Note that the temperature is different from Maxpar X8 metal labeling.
?. [Polymer loading and wash 1]
Label a 3 kDa filter and add 100 μL L buffer.
?. [Polymer loading and wash 1]
Retrieve the metal-loaded polymer mixture and then transfer all contents (approximately 100 μL) to the 3 kDa filter containing L buffer.
?. [Polymer loading and wash 1]
Add 100 μL L buffer to the polymer tube, mix thoroughly by pipetting to wash the sides of the tube, and then transfer all contents (approximately 100 μL) of the wash mixture to the 3 kDa filter. The filter should now contain approximately 300 μL of L buffer-metal-loaded polymer solution.
?. [Polymer loading and wash 1]
Use a P100 pipette to mix thoroughly, being careful not to touch the delicate filter.
?. [Polymer loading and wash 1]
Centrifuge at 12,000 × g for 25 minutes at room temperature.
?. [Antibody wash 1]
Roughly 10 minutes before MCP9 polymer centrifugation is complete, label a 50 kDa filter and add 100 μg antibody and R buffer such that the total volume is 400 μL. If the required antibody volume is >400 μL, concentrate it 15 minutes earlier by centrifuging in the filter at 12000 × g for 10 minutes at room temperature.
?. [Antibody wash 1]
Centrifuge at 12000 × g for 10 minutes at room temperature.
?. [Polymer wash 2]
Aspirate the flow-through from the bottom of the tube containing the 3 kDa filter with the metal-loaded MCP9 polymer.
?. [Polymer wash 2]
Add 300 μL of L buffer and centrifuge at 12000 × g for 30 minutes at room temperature.
?. [Antibody washes 2 and 3 and reduction]
Aspirate the flow-through from the bottom of the tube containing the 50 kDa filter with the antibody.
?. [Antibody washes 2 and 3 and reduction]
Add 400 μL of R buffer and centrifuge at 12000 × g for 10 minutes at room temperature.
?. [Antibody washes 2 and 3 and reduction]
Aspirate the flow-through from the bottom of the tube.
?. [Antibody washes 2 and 3 and reduction]
Add 400 μL of R buffer and centrifuge at 12000 × g for 10 minutes at room temperature.
?. [Antibody washes 2 and 3 and reduction]
Prepare fresh 4 mM TCEP solution by diluting 8 μL of 0.5 M TCEP stock with 992 μL of R buffer.
?. [Antibody washes 2 and 3 and reduction]
Add 100 μL of the 4 mM TCEP solution and mix quickly and thoroughly by pipetting.
?. [Antibody washes 2 and 3 and reduction]
Immediately incubate at 37° C for 30 minutes in a water bath.
?. [Polymer wash 3]
Aspirate the flow-through from the bottom of the tube containing the 3 kDa filter with the metal-loaded MCP9 polymer.
?. [Polymer wash 3]
Add 400 μL of C buffer and centrifuge at 12000 × g for 45 minutes at room temperature.
?. [Reduced antibody washes 1 and 2]
After the 30 minute antibody reduction is complete, retrieve the 50 kDa filter containing the partially reduced antibody from the 37° C water bath.
?. [Reduced antibody washes 1 and 2]
Add 300 μL of C buffer and gently mix by pipetting.
?. [Reduced antibody washes 1 and 2]
Aspirate the flow-through.
?. [Reduced antibody washes 1 and 2]
Add 400 μL of C buffer and centrifuge at 12000 × g for 10 minutes at room temperature. Centrifugation should finish slightly after polymer wash 3.
?. [Reduced antibody washes 1 and 2]
Centrifuge at 12000 × g for 10 minutes at room temperature.
?. [Conjugation]
Retrieve the 3 kDa filter unit containing the purified metal-loaded polymer.
?. [Conjugation]
Retrieve the 50 kDa filter unit containing the purified partially reduced antibody and aspirate the flow-through.
?. [Conjugation]
Resuspend the metal-loaded polymer (residual volume approximately 20 μL) in 60 μL of C buffer (total volume approximately 80 μL).
?. [Conjugation]
Transfer the resuspended contents to the corresponding partially reduced antibody in the 50 kDa filter (final conjugation volume approximately 100 μL).
?. [Conjugation]
Mix gently by pipetting.
?. [Conjugation]
Incubate at 37° C for 90 minutes in the water bath. During the incubation, label a new 100 kDa filter unit.
?. [Conjugated antibody washing]
Retrieve the 50 kDa filter unit from the water bath, and then add 200 μL of W buffer to the 50 kDa filter containing 100 μL antibody conjugation mixture (total volume approximately 300 μL).
?. [Conjugated antibody washing]
Mix gently by pipetting, and then transfer contents to the 100 kDa filter unit.
?. [Conjugated antibody washing]
Add another 100 μL of W buffer to the 50 kDa filter, mix gently by pipetting to rinse the filter, and then transfer all contents to the 100 kDa filter.
?. [Conjugated antibody washing]
Centrifuge the 100 kDa filter unit at 5000 × g for 10 minutes. If fluid fails to flow through the filter, verify the correct orientation of the filter in the centrifuge and try again, or centrifuge the mixture at 8000 × g for all wash steps.
?. [Conjugated antibody washing]
Aspirate the flow-through.
?. [Conjugated antibody washing]
Add 400 μL of W buffer, centrifuge at 5000 × g for 10 minutes, and then discard the flow-through.
?. [Conjugated antibody washing]
Add 400 μL of W buffer, centrifuge at 5000 × g for 10 minutes, and then discard the flow-through.
?. [Conjugated antibody washing]
Add 400 μL of W buffer, centrifuge at 5000 × g for 10 minutes, and then discard the flow-through.
?. [Quantitation]
Add approximately 75 μL of W buffer to the 100 kDa filter to dilute the conjugate (approximate volume of 25 μL) to a  total volume of 100 μL. Pipet to mix and carefully rinse the walls of the filter, ensuring that the pipette tip does not touch the delicate filter membrane.
?. [Quantitation]
Quantify the conjugated antibody by using a spectrophotometer to measure the absorbance of a 2 μL aliquot at 280 nm against a W buffer blank.
?. [Quantitation]
Centrifuge the 100 kDa filter at 12,000 × g for 5 minutes to remove the W buffer.
?. [Recovery and storage]
Calculate the volume of HRP-Protector (antibody stabilization buffer without sodium azide) required to obtain a final concentration of 0.5 mg/mL of conjugated antibody, or that yields a solution that is at least 50% HRP-Protector by volume.
?. [Recovery and storage]
Add the calculated volume of HRP-Protector minus the residual volume (approximately 25 μL) to the 100 kDa filter to obtain a final concentration of 0.5 mg/mL of conjugated antibody. Pipet to mix and carefully rinse the walls of the  filter, ensuring that the pipette tip does not touch the delicate filter membrane.
?. [Recovery and storage]
Label a new collection tube, invert the 100 kDa filter over to the clean collection tube, and then centrifuge the inverted filter/collection tube assembly at 1000 × g for 2 minutes.
?. [Recovery and storage]
Transfer the conjugated antibody to a labeled storage tube and store at 4° C until ready to titrate.