TITLE: Metal-antibody MIBItag conjugation kit
AUTHORS: Marc Bosse, Sean Bendall, Mike Angelo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a "protocol IO" extended version of MIBItag conjugation kit protocol from Ionpath.  </div></div>

[STEPS]
?. [Antibody buffer exchange]
Add 300 µL of Buffer 2 ( 100 mM phosphate buffer, 2.5 mM EDTA, pH 7.2) to a 50-kDa MWCO micro-filter device
?. [Antibody buffer exchange]
If step 2 not performed and antibody solution volume is less than Add the 100 µg antibody solution volume
200 µl
?. [Antibody buffer exchange]
or
Centrifuge: 12000 1324
Centrifuge: 21000 1324, 5 min
?. [Optional antibody concentration]
Critical: the 50-kDa MWCO micro-filter device is designed to hold up to 500 µLIf 100 µg of antibody requires addition of more than 200 µL in volumeAdd up to    of antibody solution  in the 50-kDa MWCO micro-filter deviceCentrifuge   or
500 µl
Centrifuge: 12000 1324
Centrifuge: 21000 1324, 5 min
?. [Antibody buffer exchange]
Add 400 µL of buffer 2 IonPath
?. [Worksheet antibody information]
ABCDEFGHIJ1Antibody (clone)Isotope/ElementPre-label Nanodrop (µg/mL)Volume to label 200 µgPost-label Nanodrop (µg/mL)Labelled volumeDilution for 200 µg/mLTotal volumeVolume of stabilizerConjug ID234567
ABCDEFGHIJ1Antibody (clone)Isotope/ElementPre-label Nanodrop (µg/mL)Volume to label 200 µgPost-label Nanodrop (µg/mL)Labelled volumeDilution for 200 µg/mLTotal volumeVolume of stabilizerConjug ID234567
?. [Antibody buffer exchange]
or  Make sure that the residual dead volume is below the bottom end of the filter (~20 µL)Do not measure with a pipette, just inspect visually if the level of the fluid is as shown in the pictures (blue dye was used for demonstration only to help visualizing the fluid)
Centrifuge: 12000 34
Centrifuge: 21000 1324, 5 min
?. [Antibody buffer exchange]
Dilute   of TCEP (0.5M) in  of buffer 2 (  final)Note:   TCEP is aliquots are stored at
8 µl
992 µl
10 µl
-20 °C
?. [Antibody reduction]
Add  of diluted TCEP to the concentrated antibody in the 50-kDa MWCO micro-filter device
100 µl
?. [Antibody reduction]
Mix antibody and TCEP solution by flicking the 50-kDa MWCO micro-filter device gently
?. [Antibody reduction]
Incubate at  for  ONLYDo not incubate longer than 30 min, this will result in full reduction of disulfide bonds necessary for the structural integrity of the antibody
37 °C
?. [Antibody wash]
After incubation, add immediately  of buffer 3 to the partially reduced antibody in the50-kDa MWCO micro-filter device
300 µl
?. [Antibody wash]
Mix reduced antibody and wash buffer 3 (Tris buffed salt, 1 mM EDTA, pH 7.5) by flicking the 50-kDa MWCO micro-filter device gently
?. [Antibody wash]
or
Centrifuge: 12000 1324, 10 min
Centrifuge: 21000 1324, 5 min
?. [Antibody wash]
Discard the flow through
?. [Antibody wash]
Add additional  of buffer 3
400 µl
[once]
?. [Conjugation of metal and antibody]
Reconstitute preloaded polymer in   of buffer 3
100 µl
?. [Conjugation of metal and antibody]
Transfer the preloaded polymer solution in the  reduced antibody/ 50-kDa MWCO micro-filter device
?. [Conjugation of metal and antibody]
Mix reduced antibody and preloaded polymer by flicking the 50-kDa MWCO micro-filter device gently
?. [Conjugation of metal and antibody]
Inccubate  for   to
37 °C
?. [Final wash]
Add 250 µL of buffer 4 to the antibody conjugation mixture in the the 50-kDa MWCO micro-filter device
?. [Optional antibody concentration]
Make sure to align the filters parallel to centrifuge force to ensure optimal tengential flow filtration of both filtersThis can be done by centering the hinge of the collecting tube between the two filtersThe 50-kDa MWCO micro-filter device is then placed in the centrifuge with the hinge outward
?. [Final wash]
Mix antibody conjugation mixture and buffer 4 (Tris buffed salt, pH 7.5) by flicking the 50-kDa MWCO micro-filter device gently
?. [Final wash]
or
Centrifuge: 12000 1324, 10 min
Centrifuge: 21000 1324, 5 min
?. [Final wash]
Discard the flow through
?. [Final wash]
Add  of buffer 4 in the the 50-kDa MWCO micro-filter device to 24
400 µl
?. [Final wash]
Add  of buffer 4 in the the 50-kDa MWCO micro-filter device to 24
400 µl
?. [Final wash]
Add  of buffer 4 in the the 50-kDa MWCO micro-filter device to 24
400 µl
?. [Final wash]
Add  of buffer 4 in the the 50-kDa MWCO micro-filter device to 24
400 µl
?. [Labelled antibody retrieval]
Add  of buffer 4 to the washed labelled antibody in the the 50-kDa MWCO micro-filter device
50 µl
?. [Labelled antibody retrieval]
Mix washed labelled antibody and buffer 4 by flicking the 50-kDa MWCO micro-filter device gently
?. [Labelled antibody retrieval]
Invert the micro-filter into a new collection tube
?. [Labelled antibody retrieval]
Centrifuge: 10000 1324, 1 min
?. [Labelled antibody retrieval]
Add  of buffer 4 to the washed labelled antibody in the the 50-kDa MWCO micro-filter device  to 33
80 µl
?. [Labelled antibody retrieval]
Measure the volume retrieved with a pipette (200 µL) and register data in labelled volume on the worksheet
?. [Labelled antibody retrieval]
Open Nanodrop using Protein 280 and chose type = IgG
?. [Labelled antibody retrieval]
Blank with buffer 4
?. [Labelled antibody retrieval]
Take  of labelled antibody and measure concentration
1.5 µl
?. [Labelled antibody retrieval]
Clean the cell with ultrapure (type 1) water
?. [Labelled antibody retrieval]
Register data in labelled volume on the worksheet
?. [MIBItracker database/storage formulation]
Open MIBItracker and click on CONJUGATES tab
?. [MIBItracker database/storage formulation]
Click on Add Conjugate tab
?. [MIBItracker database/storage formulation]
Select the antibody reference used for the conjugation Fill the following fields:  Isotope/ Channel; Element; µg for labelling; Responsible
?. [MIBItracker database/storage formulation]
Click on SUBMIT, this will generate a conjugation ID numberClick on back to Conjugate and filter by conjugation date
?. [MIBItracker database/storage formulation]
Record the ID number on your experiment worksheet
?. [MIBItracker database/storage formulation]
Click on Edit Conjugate
?. [MIBItracker database/storage formulation]
Enter Nanodrop reading post-labelling and Volume of labeled antibody in the respective fields
?. [MIBItracker database/storage formulation]
For a standard labeled stock concentration (200 µg/mL), the Dilution factor and the volume of Antibody Stabilizer is calculated automatically If the Dilution factor is less than
?. [MIBItracker database/storage formulation]
Add the volume of Antibody Stabilizer to the antibody labeled collection tube. Use Tris-based antibody stabilizer (Boca Scientific).
?. [MIBItracker database/storage formulation]
Transfer the diluted labeled antibody into a new screw cap microcentrifuge tube
?. [MIBItracker database/storage formulation]
Label the tube with printer Brady 21-Lab, (Label for 1.5 mL vial, Auto bold)Line1: Target name, ChannelLine2: ID: numberLine3: date (mm/dd/yy)
?. [MIBItracker database/storage formulation]
Hand write the target name and the channel on a dot label and place on the screw cap
?. [Short term storage at 4˚C/ Long term storage Lyophilized]
Labeled can be stored short term for Preferably, the newly labeled labeled antibody is immediately placed in the lyophilization box at 4˚Cin queue to be lyophilized and then placed at 4˚C in designated box for longterm storage management