May 16, 2022
MSD Gold Streptavidin Antibody Preparation and Plate Run Protocol
- Kamaljot Gill1,2,
- Maria Sckaff3,
- Claire D Clelland2
- 1Gladstone Institutes, San Francisco, CA, United States;
- 2University of California, San Francisco, Weill Institute for Neurosciences, San Francisco, CA, United States;
- 3University of California, San Francisco
Protocol Citation: Kamaljot Gill, Maria Sckaff, Claire D Clelland 2022. MSD Gold Streptavidin Antibody Preparation and Plate Run Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvobkj9l4o/v1
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: March 25, 2022
Last Modified: May 16, 2022
Protocol Integer ID: 59888
Keywords: Buffer Exchange, Biotinylate the Antibody, Sulfo-tag the Antibody, MSD, ELISA, protein quantification, streptavidin, antibody, protein
Abstract
This protocol describes how to conjugate antibodies and run the Meso Scale Discovery (MSD) Sandwich enzyme-linked immunosorbent assay (ELISA) on MSD GOLD 96-well Small Spot Streptavidin SECTOR Plates. This protocol is adapted from MSD GOLD™ Streptavidin Plate and Avidin Plates Quick Guide 1 and MSD GOLD™ SULFO-TAG NHS-Ester Conjugation Quick Guide 2 and optimized for C9orf72 dipeptide repeat detection from human iPSC derived neurons.
Attachments
Guidelines
References
1MSD GOLD™ SULFO-TAG NHS-Ester Conjugation Quick Guide
2MSD® Biotin Conjugation Quick Guide
3MSD® GOLD Streptavidin and Avidin Plates Quick Guide
Materials
Reagents List
| A | B | C | |
| Reagents | Manufacturer | Catalog Number | |
| MSD GOLD SULFO-TAG NHS-Ester Conjugation Pack | MSD | R31AA | |
| Zeba Spin Desalting Columns | ThermoFisher | 89882 | |
| EZ-LinkTM Sulfo-NHS-LC-Biotin, No-WeighTM Format | ThermoFisher | A39257 | |
| MSD GOLD 96-well Small Spot Streptavidin SECTOR Plate | MSD | L45SA | |
| Blocker A | MSD | R93BA | |
| MSD GOLD Read Buffer A | MSD | R92TG | |
| PBS, pH 7.4 | ThermoFisher | 10010023 | |
| Tween-20 | Sigma | P1379 | |
| Nuclease-Free Water (not DEPC-Treated) | ThermoFisher | AM9932 |
MSD GOLD SULFO-TAG NHS-Ester Conjugation PackMESO SCALE DIAGNOSTICS, LLC.Catalog #R31AA
Zeba™ Spin Desalting Columns, 7K MWCO, 0.5 mLThermo FisherCatalog #89882
EZ-Link™ Sulfo-NHS-LC-Biotin, No-Weigh™ FormatThermo FisherCatalog #A39257
MSD GOLD 96-well Small Spot Streptavidin SECTOR PlateMESO SCALE DIAGNOSTICS, LLC.Catalog #L45SA
Blocker AMESO SCALE DIAGNOSTICS, LLC.Catalog #R93BA
MSD GOLD Read Buffer AMESO SCALE DIAGNOSTICS, LLC.Catalog #R92TG
PBS pH 7.4Thermo Fisher ScientificCatalog #10010023
Tween 20SigmaCatalog #P1379
Nuclease-Free Water (not DEPC-Treated)Thermo FisherCatalog #AM9932
Alternative Reagents List
| A | B | C | D | |
| Reagents | Alternative Reagents | Manufacturer | Catalog Number | |
| MSD Conjugation Buffer | PBS, pH 7.4 | ThermoFisher | 10010023 | |
| MSD Conjugation Storage Buffer | PBS, 0.05% Sodium Azide | Teknova | P0202 | |
| Blocker A | BSA | Sigma | A4503 |
PBS 0.05% Sodium AzideTeknovaCatalog #P0202
Bovine Serum AlbuminMillipore SigmaCatalog #A4503
Equipment List
| A | B | C | |
| Equipment | Manufacturer | Catalog Number | |
| Microseal 'B' PCR Plate Sealing Film, adhesive, optical | BioRad | MSB1001 | |
| 1.5 mL Eppendorf tubes | Fisher Scientific | 14-666-321 | |
| Meso Scale Discovery (MSD) Model 1250 Sector Imager 2400 | MSD | 1250 | |
| HeidolphTM Titramax Vibrating Platform Shakers | Fisher Scientific | 13-889-420 | |
| Centrifuge (capable of 1,500g) | Any | Any | |
| Vortex | Any | Any |
Equipment
Microseal 'B' PCR Plate Sealing Film, adhesive, optical
NAME
Plate Sealing Film
TYPE
Microseal
BRAND
MSB1001
SKU
LINK
Equipment
Microcentrifuge Tubes with Locking Snap Cap
NAME
Microcentrifuge Tubes
TYPE
Fisherbrand™
BRAND
14-666-321
SKU
LINK
Equipment
Meso Scale Discovery (MSD) Model 1250 Sector Imager 2400
NAME
multiplex assay reader
TYPE
MESO SECTOR
BRAND
1250
SKU
LINK
Equipment
Heidolph™ Titramax Vibrating Platform Shakers
NAME
Shaker
TYPE
Heidolph™
BRAND
13-889-420
SKU
LINK
Buffer Exchange the Antibodies
Buffer Exchange the Antibodies
20m
20m
Note
It is only needed to buffer exchange your antibodies if they are in buffers with preservatives such as sodium azide or EDTA or in buffers that contain primary amines or glycerol.
Chill PBS (or MSD Conjugation Buffer) and ultrapure water On ice .
Equilibrate Zeba Spin Desalting Columns, MSD Storage Buffer, and Sulfo-NHS-LC-Biotin at Room temperature .
Use one Zeba column per 70 µL of antibody.
In order to both biotinylate and sulfo-tag the antibody, you need at least 140 µL of antibody at an optimal concentration of 1.0 mg/mL .
It still possible to move forward with a less concentrated sample.
Dilute the antibodies with ice-cold PBS if necessary.
Remove the Zeba columns' bottom closure and loosen the cap.
Note
Do not remove the cap.
Place the column in a collection tube to remove the storage buffer.
Spin at 1500 x g, 00:01:00 . Empty collection tube.
1m
Wash 1: Add 300 µL of PBS (or MSD Conjugation Buffer) to the column. Spin at 1500 x g, 00:01:00 . Empty collection tube.
1m
Wash 2: Add 300 µL of PBS (or MSD Conjugation Buffer) to the column. Spin at 1500 x g, 00:01:00 . Empty collection tube.
1m
Wash 3: Add 300 µL of PBS (or MSD Conjugation Buffer) to the column. Spin at 1500 x g, 00:03:00 . Empty collection tube.
3m
Change collection tube to a clean Eppendorf tube for sample recovery. Label one Eppendorf tube/sample for each sample to be biotinylated or sulfo-tagged.
Pipette 70 µL of the antibody to the spin column.
Spin at 1500 x g for 3-4 minutes.
Save the eluent On ice .
Note
The eluent is the buffer exchanged antibody.
Biotinylate the Antibody
Biotinylate the Antibody
2h
2h
Note
Note on planning when to biotinylate your antibodies: Before performing this step, plan to tag multiple antibodies at the same time to save both money and reagents.
Calculate how much Sulfo-NHS-LC-Biotin you need per antibody, using the following formula:
- 1,000 × ([Concentration of antibody in mg/mL]/ 150,000 Da) × 20 × 70 µL of antibody = nmol of Biotin needed
- This nmol of Biotin needed divided by 0.5 nmol/μL Biotin reagent = μL of Sulfo-NHS-LC Biotin needed
- See attached examples at the end of this document
Add 180 µL of ultrapure H2O to the 1 mg vial to Sulfo-NHS-LC-Biotin.
Dilute the Sulfo-NHS-LC-Biotin by adding 10 µL of the stock to 190 µL of cold water. Once formed, this is highly unstable and should be used immediately.
Add the calculated volume of diluted reconstituted Sulfo-NHS-LC-Biotin to each antibody.
Let the antibody and biotin incubate at Room temperature for 02:00:00 in the dark.
2h
Sulfo-tag the Antibody
Sulfo-tag the Antibody
2h
2h
Note
Note on planning when to sulfo-tag: Before performing this step, plan to tag multiple antibodies at the same time to save both money and reagents.
Calculate how much SULFO-TAG NHS-Ester you need per antibody, using the following formula:
- 1,000 × ([Concentration of antibody in mg/mL]/ 150,000 Da) × 20 × 70 µL of antibody = nmol of Sulfo-Tag reagent needed
- This nmol of Sulfo-Tag needed divided by 3.0 nmol/µL = µL of sulfo-tag ester solution needed
- See attached examples at the end of this document
Add 50 µL of ice-cold ultrapure H2O to the 150 nmol SULFO-TAG NHS-Ester Vial to make a 3 nmol/μL solution. Once formed, this is highly unstable and should be disposed.
Vortex the solution lightly.
Add the calculated volume of diluted reconstituted SULFO-TAG NHS-Ester reagent to each antibody.
Let the antibody and SULFO-TAG NHS-Ester incubate at Room temperature for 02:00:00 in the dark.
2h
Clean-up the Biotinylated Antibody
Clean-up the Biotinylated Antibody
15m
15m
Equilibrate Zeba Spin Desalting Columns, MSD Storage Buffer at Room temperature .
Place the column in a collection tube to remove the storage buffer.
Remove the columns' bottom closure and loosen the cap
Note
Do not remove the cap.
Spin at 1500 x g, 00:01:00 . Empty collection tube.
1m
Wash 1: Add 300 µL of MSD Conjugate Storage Buffer to the column. Spin at 1500 x g, 00:01:00 . Empty collection tube.
1m
Wash 2: Add 300 µL of MSD Conjugate Storage Buffer to the column. Spin at 1500 x g, 00:01:00 . Empty collection tube.
1m
Wash 3: Add 300 µL of MSD Conjugate Storage Buffer to the column. Spin at 1500 x g, 00:03:00 . Empty collection tube.
3m
Change collection tube for sample recovery.
Pipette 70 µL of the unpurified biotinylated antibody to the spin column.
Spin at 1500 x g for 3-4 minutes.
Save the eluent On ice .
Note
The eluent is the biotinylated antibody. It is stable at 4 °C for 1 year.
Cleanup the Sulfo-Tagged Antibody
Cleanup the Sulfo-Tagged Antibody
15m
15m
Equilibrate Zeba Spin Desalting Columns, MSD Storage Buffer at Room temperature .
Remove the columns' bottom closure and loosen the cap.
Note
Do not remove the cap.
Place the column in a collection tube to remove the storage buffer.
Spin at 1500 x g, 00:01:00 . Empty collection tube.
1m
Wash 1: Add 300 µL of MSD Conjugate Storage Buffer to the column. Spin at 1500 x g, 00:01:00 . Empty collection tube.
1m
Wash 2: Add 300 µL of MSD Conjugate Storage Buffer to the column. Spin at 1500 x g, 00:01:00 . Empty collection tube.
1m
Wash 3: Add 300 µL of MSD Conjugate Storage Buffer to the column. Spin at 1500 x g, 00:03:00 . Empty collection tube.
3m
Change collection tube for sample recovery.
Pipette 70 µL of the unpurified SULFO-TAG NHS-Ester tagged antibody to the spin column.
Spin at 1500 x g for 3-4 minutes.
Save the eluent On ice .
Note
This is the SULFO-TAG NHS-Ester tagged antibody. It is stable at 4 °C for 1 year.
Day 1 of MSD: Coating the Plate with Capture Antibody
Day 1 of MSD: Coating the Plate with Capture Antibody
30m
30m
Dilute biotinylated capture antibodies in 1x DPBS to your desired concentration.
Add 30 µL of the diluted antibody to the corner of each well of the MSD 96 Streptavidin Small. Spot Plate according to the plate format.
Note
IMPORTANT: From this point forward, never let the plate dry.
Tap the plate on its edges to ensure the antibody is spread evenly across the well.
Seal the plate with parafilm to avoid loss of antibody due to evaporation.
Store in the plate at 4 °C to incubate Overnight without shaking.
Day 2 of MSD
Day 2 of MSD
5h
5h
Tap out the plate to dispose of the capture antibody.
Add 150 µL /well of Blocking Solution (3% Blocker A (or BSA) + PBS) per well.
| A | B | |
| 3% Blocker A in 1x PBS (Store at 4°C) | ||
| For 100 mL | ||
| Blocker A | 3 g | |
| 1X PBS | 100 mL | |
| (Stir or shake overnight at 4°C to make sure it is dissolved completely.) | ||
Seal the plate and incubate at Room temperature with shaking at 750 rpm, 01:00:00 .
1h
Prepare the lysate samples according to plate layout, by diluting the protein samples in the lysate buffer to the desired lysate concentrations.
Tap out the plate.
Wash 1x with 150 µL /well of PBS - T (0.05% Tween).
Discard the wash solution, without letting the plate dry.
Add 25 µL /well of the lysate to the target wells.
Note
Add lysate to the bottom corner of the wells.
Seal the plate and incubate at Room temperature with shaking at 750 rpm for 1-2 hours.
2h
While the lysates incubate, prepare the detection antibodies (your sulfo-tagged antibodies) in 1% MSD Blocker A in 1x DPBS.
Discard the lysate solution.
Note
Do not let the plate dry, but remove the excess solution.
Wash with PBS - T (0.05% Tween).
Wash with 150 µL /well of PBS - T (0.05% Tween). (1/3)
Wash with 150 µL /well of PBS - T (0.05% Tween). (2/3)
Wash with 150 µL /well of PBS - T (0.05% Tween). (3/3)
Discard the wash solution, but do not let the plate dry.
Add 25 µL /well of sulfo-tag antibodies (in 1% Blocker A in DPBS) to the plate layout.
Seal the plate and incubate at Room temperature with shaking at 750 rpm, 01:00:00 .
1h
Tap out the plate.
Wash with PBS - T (0.05% Tween).
Wash with 150 µL /well of PBS - T (0.05% Tween). (1/3)
Wash with 150 µL /well of PBS - T (0.05% Tween). (2/3)
Wash with 150 µL /well of PBS - T (0.05% Tween). (3/3)
Tap out the plate.
Add 150 µL /well of Read Buffer A using reverse pipetting to avoid making bubbles.
Note
IMPORTANT: Ensure there is no plastic wrap, tape, or parafilm on the plate.
Read the plate immediately.
5m
Sample calculations
Sample calculations
Note
This section outlines sample calculations for the biotinylation and sulfo tagging of antibodies for the lVISD assay, as described in the protocol above.
Relevant notes, using Poly-GR 1VIABN778 and Poly-PR ABN1354 antibodies as references:
- 0.5mg/mL is the concentration of the antibody
- 150,000Da is the protein weight for IgG protein
- 20:1 is the challenge ratio
- 70 pL is the volume of the protein solution
Sample Biotinylation Calculations
Sample Biotinylation Calculations
Poly-GR MABN778 and Poly-PR ABN1354
4.67 nmol of sulfo-NHS-LC Biotin required
9.34 µL of sulfo-NHS-LC Biotin stock solution .
Poly-GA MABN889
3.08 nmol of sulfo-NHS-LC Biotin required
6.16 µL of sulfo-NHS-LC Biotin stock solution.
Sample Sulfo-tag Calculations
Sample Sulfo-tag Calculations
Poly-GR MABN778 and Poly-PR ABN1354
4.67 nmol of sulfo-tag reagent required.
1.56 µL of sulfo-tag ester solution .
Poly-GA MABN889
3.08 nmol of sulfo-tag reagent required.
1.03 µL of sulfo-tag ester solution.