eProtein Quantification Kit Protocol

General Information

The quantificationkit protcol allows the user to quantify the protein produced in the scale-up as it was quantified on cartridge.

Features and benefit

Contents

Component	Volume	Cap Color	Storage Temperature	Tube Reference number
Detector Protein	75 µL		-80°C
Universal Control	20 µL		-80°C
Complementation Control	20 µL		-80°C
Wash buffer	800 µL		-80°C

User supplied equipment

▷ Microplate reader capable of measuring fluorescence at Ex/Em = 485/520.
▷ Assay plate - low volume, 384-well, black/clear bottom, polystyrene nonbinding surface microplate (Corning, 3544).
▷ Adhesive PCR plate seal.

Storage and Stability

▷ The kit should be stored at −80°C for 1 year from receipt.
▷ Avoid repeated freeze-thaw cycles.
▷ Thaw reagents on ice, briefly centrifuge, and pipette mix prior to use.
▷ Reactions should be assembled on ice.

Reaction Assembly Overview

	Standards	Neg. Control	Pos. Control	Samples
Wash Buffer	8 µL	9 µL	8 µL	5 µL
Diluted Uni. Control	4 µL	X	X	X
Comp. Control	X	X	1 µL	X
Protein Sample	X	X	X	4 µL
Detector Protein	X	3 µL	3 µL	3 µL
Total per Well	12 µL	12 µL	12 µL	12 µL

Overview of reaction assembly, see proceeding points for detailed protocol.

Standard Curve Preparation

1. Prepare the following serial dilution of Universal Control.

• Prepare a fresh set of standards for each quantification.
• Pipette mix five times for each dilution step.
• Use a fresh pipette tip for each dilution step.
• Each dilution provides enough standard to set up triplicate readings.

Standard Concentration	Volume of Universal Control	Volume of Wash Buffer	Dilution Number
18 µM	15 µL of undiluted	15 µL	1
9 µM	15 µL of 18 µM dilution	15 µL	2
4.5 µM	15 µL of 9 µM dilution	15 µL	3
2.25 µM	15 µL of 4.5 µM dilution	15 µL	4
0 µM	0 µL	15 µL	5

2. Add 8 µL of Wash Buffer to five wells of the assay plate in triplicate .
3. Add 4 µL of each standard in triplicate to the wells containing Wash Buffer (total well volume of 12 µL).

Preparation of Negative Control Reactions

1. Add 9 µL of Wash Buffer in triplicate to the assay plate.
2. Add 3 µL of Detector Protein to the three wells containing Wash Buffer (total well volume of 12 µL).

Preparation of Positive Control Reactions

1. Add 8 µL of Wash Buffer in triplicate to the assay plate.
2. Add 1 µL of Complementation Control to the wells containing Wash Buffer.
3. Add 3 µL of Detector Protein to the three wells containing Wash Buffer and Complementation Control (total well volume of 12 µL).

Preparation of Test Samples

1. For each sample to be quantified, add 5 µL of Wash Buffer in triplicate to the assay plate.
2. Add 4 µL of sample protein to the wells containing Wash Buffer.

• Proteins with a predicted yield of more than 18 µM on the eProtein DiscoveryTM should be diluted 1 in 2 before quantification.

3. Add 3 µL of Detector Protein to each well containing Wash Buffer and sample protein (total well volume of 12 µL).

Assay Plate Layout Overview

Incubation

1. Seal the assay plate with an adhesive PCR plate seal. Ensure the plate is fully sealed before proceeding.
2. Briefly vortex the plate and pulse centrifuge for 15 secs.
3. Incubate the assay plate for 5 hours at 29°C.

• For overnight quantification, the assay plate can be incubated at 29°C directly in the plate reader with the program set to measure fluorescence after 5 hours. Ensure that the plate seal is left on and the program is set to bottom reading mode.

Measurement

1. After 5 hours measure the fluorescence of all standards, controls, and samples in a plate reader with a 485 nm excitation filter and a 520 nm emission filter.

Data Analysis

1. Calculate the average fluorescence reading for each standard.
2. Subtract the 0 µM reading from all other standard readings.
3. Plot the µM concentration of each standard against the measured fluorescence intensity, fit a linear trendline, and set the intercept to 0.
4. Calculate the average fluorescence intensity for each set of controls and protein samples.
5. Subtract the average fluorescence reading for the negative control from the positive control and protein sample measurements.
6. Use the linear trendline equation to calculate the molarity of the protein samples and positive control.

• If the protein was diluted prior to reaction assembly (e.g. if the predicted concentration was above 18 µM), multiply by the dilution factor to calculate the concentration of the original sample.