Aug 07, 2019
Version 2
CODEX Oligo-labeled Antibody Conjugation V.2
- Yury Goltsev1,
- Nikolay Samusik1,
- Julia Kennedy-Darling1,
- Salil Bhate1,
- Matthew Hale1,
- Gustavo Vazquez1,
- Sarah Black1,
- Garry Nolan1
- 1Stanford University
- Human Cell Atlas Method Development Community
External link: https://www.cell.com/cell/pdf/S0092-8674(18)30904-8.pdf
Protocol Citation: Yury Goltsev, Nikolay Samusik, Julia Kennedy-Darling, Salil Bhate, Matthew Hale, Gustavo Vazquez, Sarah Black, Garry Nolan 2019. CODEX Oligo-labeled Antibody Conjugation. protocols.io https://dx.doi.org/10.17504/protocols.io.3fugjnw
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 28, 2019
Last Modified: August 07, 2019
Protocol Integer ID: 23764
Abstract
CODEX is a technology that uses oligo labeled antibodies, specialized fluorescent probes, and a companion instrument along-side a standard fluorescence microscope to create single-cell resolution fluorescence data across a multitude of parameters within spatial context in a single tissue. CODEX was developed by Yury Goltsev and Nikolay Samusik in the laboratory of Garry Nolan at Stanford. The technology is being commercialized by Akoya Biosciences.
Guidelines
Purified antibody stock can not be stored with any carrier protein or glycerol. Request the vendor to provide purified antibody in a BSA-free form.
A pre-purification process must be performed if a BSA-free antibody stock cannot be found.
Measure concentration of purified antibody with nanodrop before conjugation procedure. Concentrations on the tube label are not always accurate.
Materials
| PRODUCT | PROVIDER | CATALOG NUMBER | |
| 50KDa MWCO filter | Millipore | UFC505096 | |
| Antibody Reduction Solution 1 | Akoya | RGT2004EA | |
| Antibody Reduction Solution 2 | Akoya | RGT2004EA | |
| Antibody Conjugation Solution | Akoya | RGT2004EA | |
| Filter Blocking Solution | Akoya | RGT2004EA | |
| CODEX Antibody Tags | Akoya | RGT2004EA | |
| Antibody Purification Solution | Akoya | RGT2004EA | |
| Antibody Storage Solution | Akoya | RGT2004EA | |
| Nanodrop 2000c | ThermoFisher | ND-2000c | |
| Eppendorf microcentrifuge | Eppendorf | 5418 |
Antibody Disulfide Reduction Reaction
Antibody Disulfide Reduction Reaction
Retrieve one MWCO filter column for each antibody to be conjugated. Block nonspecific antibody binding to the MWCO filter columns by adding 500ul Filter Blocking Solution to the top of each column and spinning down at 12,000g for 2 minutes.
12000 x g
00:02:00
Room temperature
Aspirate all liquid from the top of each MWCO filter column and discard flow-through in the collection tube. Exercise caution and do not scrape and damage the filter with the aspirator tip.
Measure concentration of stock purified antibody by nanodrop with pre-set IgG settings after blanking against the buffer they are suspended in, usually PBS. Calculate the volume needed for 50ug of antibody based on the nanodrop measurement.
Add 50ug of antibody to the top of each MWCO filter column and spin down at 12,000g for 8 minutes. The resulting solution should be 50ug of antibody concentrated into approximately 25ul.
12000 x g
00:08:00
Room temperature
Discard flow-through.
Prepare Antibody Reduction Master Mix by combining Antibody Reduction Solution 1 with Antibody Reduction Solution 2.
20ul of Antibody Reduction Solution 1 is needed for every 3 antibodies to conjugate.
20ul of Antibody Reduction Solution 1 is mixed with 825ul of Antibody Reduction Solution 2.
Add 260ul Antibody Reduction Master Mix to the top of each MWCO filter column. Gently pipet up and down to mix reagent with antibody. Incubate at RT for 30 minutes.
00:30:00
Room temperature
After 30 minutes spin down the MWCO filter columns at 12,000g for 8 minutes.
12000 x g
00:08:00
Room temperature
Discard flow-through.
Antibody Conjugation Reaction
Antibody Conjugation Reaction
Add 450ul Antibody Conjugation Solution to the top of each MWCO filter column and spin down at 12,000g for 8 minutes. During this spin, prepare the antibody tags.
12000 x g
00:08:00
Room temperature
The antibody tag preparation is time sensitive and must be done immediately prior to use. The antibody tag aliquots are to be used once for every 50ug antibody conjugation reaction.
Pipet 220ul Antibody Conjugation Solution into each antibody tag aliquot. Ensure that the aliquot is dissolved with gentle pipetting.
After the spin in step 10 is completed, discard flow-through and add the dissolved antibody tag solutions from step 12 into each corresponding MWCO filter column. Pipet up and down gently to mix the reagents.
Close the MWCO filter column lids and incubate the conjugation reaction at RT for 2 hours.
02:00:00
Room temperature
After 2 hours, spin down the MWCO filter columns at 12,000g for 8 minutes.
12000 x g
00:08:00
Room temperature
Discard flow-through.
Antibody Purification and Storage
Antibody Purification and Storage
Add 450ul Antibody Purification Solution to each MWCO filter column and spin down at 12,000g for 8 minutes.
12000 x g
00:08:00
Room temperature
Discard flow-through.
Add another 450ul Antibody Purification Solution to each MWCO filter column and spin down at 12,000g for 8 minutes.
12000 x g
00:08:00
Room temperature
Discard flow-through.
Add another 450ul Antibody Purification Solution to each MWCO filter column and spin down at 12,000g for 8 minutes.
12000 x g
00:08:00
Room temperature
Discard flow-through.
Add 100ul Antibody Storage Solution to the top of each MWCO filter column. Gently pipet up and down 10 or more times and wash the sides of the filters in the column. 50ug antibody should be dissolved in 100ul Antibody Storage Solution. If the conjugation scale is larger than 50ug, add more Antibody Storage Solution according to this ratio.
Invert the filters into a fresh collection tube. Spin down at 3000g for 2 minutes. KEEP THE COLLECTED SOLUTION.
3000 x g
00:02:00
Pipet the conjugated antibody solutions into sterile screw-top tubes and store for one year at 4 °C .