Instructions

Incubation

1. Prepare the Incubation mix in a microcentrifuge tube according to the table below.

Incubation mix per ½ 96-well plate (μL)

Olink® Target 48 Incubation Solution 168

Olink® Target 48 Frw-probes 21

Olink® Target 48 Rev-probes 21

Total 210

2. Vortex and spin down the Incubation mix. Transfer 23 µL of the Incubation mix to each well of a new 8-well strip.

3. Transfer 3 μL of Incubation mix to each well of the first 6 columns of a 96-well plate by

reverse pipetting and name the

plate Incubation Plate.

4. Add 1 µL of each sample using a multi-channel pipette to the bottom of the well, 1 µl of Sample Control to the three top wells

(yellow), 1 µL of Negative Control to two wells (red), and 1 µL of Calibrators to three wells (green), according to the plate

layout.

Sample Control

Negative Control

Calibrator

5. Seal the plate with an adhesive plastic film, spin at 400 – 1000 x g, 1 min at room temperature. Incubate overnight at +4 °C.

Extension

1. Prepare an extension mix according to the table below.

Extension mix per ½ 96-well plate (µL)

High Purity Water (+4 °C) 4350

Olink® Target 48 PEA Enhancer 580

Olink® Target 48 PEA Solution 580

Olink® Target 48 PEA Enzyme 58

Total 5 568

2. Bring the Incubation Plate to room temperature, spin at 400 – 1000 x g for 1 min. Preheat the PCR machine.

3. Vortex the Extension mix and pour it into a multichannel pipette reservoir.

4. Start a timer for 5 min and transfer 96 µL of Extension mix to the upper parts of the well walls of the Incubation Plate by

using

reverse pipetting.

5. Seal the plate with a new adhesive plastic film, use the MixMate® to vortex the plate at 2500 rpm for 30 sec, ensuring that all

wells are mixed, and spin down.

Olink® Target 48

Short instructions

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® Olink® Target 48 Short instructions Incubation 1. Prepare the Incubation mix in a microcentrifuge tube according to the table below. Incubation mix per ½ 96-well plate (μL) Olink® Target 48 Incubation Solution Olink® Target 48 Frw-probes Olink® Target 48 Rev-probes Total 168 21 21 210 2. Vortex and spin down the Incubation mix. Transfer 23 µL of the Incubation mix to each well of a new 8-well strip. 3.
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6. Place the Incubation Plate in the thermal cycler and start the PEA program. (50 °C 20 min, 95 °C 5 min (95 °C 30 sec, 54 °C 1 min, 60 °C 1 min) x 17, 10 °C hold) Detection 1. Prepare and prime an Olink® 48.48 IFC for Protein Expression. Briely, inject one control line fluid syringe into each accumulator on the chip, and then prime the IFC on the Olink® Signature Q100 following the instructions on the instrument screen. 2. Thaw the Primer Plate, vortex and spin briefly. 3.