dynamic Biosensors PF-NH2-3 Amine Coupling Kit 3 for Proteins User Manual

: June 2, 2024
: dynamic BIOSENSORS

Table of Contents

Key Features
Workflow Overview
Product Description
Additional Materials Required
Important Notes
Additional Information
Useful Order Numbers
Customers Support
References
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dynamic Biosensors PF-NH2-3 Amine Coupling Kit 3 for Proteins

dynamic Biosensors PF-NH2-3 Amine Coupling Kit 3 for
Proteins

Key Features

Coupling of biomolecules with primary amines (e.g. NH2 -terminus, lysines) to DBCO-modified DNA* in a reaction tube
Convenient standard chemistry
Applicable for proteins (and peptides) (MW > 5 kDa)
Coupling of multiple proteins can be performed simultaneously
Yields >95 % pure protein-DNA conjugate with controlled quality of your product
With any DNA sequence and length up to 150 bases feasible

Workflow Overview

3-Step Conjugation Workflow (in-vitro)

Workflow Overview

DNA Modification
The DNA is activated with amine reactive groups.
Protein Conjugation
After incubation the excess linker is removed by a spin column. The protein/peptide is added to the functionalized DNA and incubated for at least 1 h.
Purification
The protein-DNA conjugate is purified using the proFIRE® system.
Ready-to-use fractions
The fractions with protein DNA conjugate are ready for further processing.

Timeline: Hands on time < 1 h | Incubation ~ 2 h | Total ~ 3 h

Product Description

Order Number PF-NH2-3

TABLE 1 | Contents and Storage Information

Material	Cap	Amount	Storage
Conjugation Buffer	trans parent	5 x 1.8 mL	-20°C
Dilution Buffer	trans parent	1.8 mL	-20°C
ddH2O	trans parent	1.5 mL	-20°C
Crosslinker	brown	5 x	-20°C
Purification spin column	red	10 x	2-8°C
2.0 mL Reaction tubes for Purification spin column		10 x	r.t.
Centrifugal filter unit (3 kDa MWCO)1		5 x	r.t.
Centrifugation collection tube		10 x	r.t.

For in vitro use only.

Please check date of expiry on the kit. Products are shipped at ambient temperature. The kit contains reagents sufficient for 5 conjugations of approx. 50-200 µg biomolecule each. The resin slurry of the Purification spin column contains 0.02 % sodium azide.

Additional Materials Required

TABLE 2 | Additional Materials.

Material	Comment
DNA	We recommend to use 3 – 4 nmol DNA (modified with DBCO, HPLC grade) for

one reaction
Benchtop microcentrifuge| Capable between 1,000 x g and 13,000 x g
Vortexer|
1.5 mL reaction tubes|
UV-Vis spectroscopy (e.g. Nanodrop)| Concentration determination of the conjugate

All necessary solutions and buffers are included in the kit.

Important Notes

Do not use any buffer containing primary amines (i.e. TRIS, glycine) during conjugation process.
Dithiothreitol (DTT) can be used up to 1 mM during the conjugation process.
Do not use 2-Mercaptoethanol or other thiol-based reducing agents during conjugation process. If a reducing agent is necessary, TCEP is recommended up to 1 mM.
Avoid using partially purified protein samples or protein samples containing carriers (e.g. BSA).
To get highest reaction yields, the ligand should be dissolved in Conjugation Buffer. Buffer exchange is recommended prior to conjugation process1.
Before you begin, briefly centrifuge all tubes with brown and transparent caps to ensure that all material is at the bottom of the tubes.
For molecules with a molecular weight around or lower than 5 kDa, special care during purification process shall be taken. A few peptides may not give a proper purification using the provided proFIRE® column. For more information please email [email protected].
If the pI of the protein is < 6, it might be necessary to use a lower pH buffer.
For more information, please emailsupport@dynamic- biosensors.com.

3-Step Conjugation of a Biomolecule to a Nano lever in a Reaction Tube

Please read the entire protocol before starting and perform conjugation without interruption.
TIP: The protocol can be performed simultaneously for multiple coupling reactions.
Before you begin: Allow the crosslinker to reach room temperature before use.

Nano lever Modification

Dissolve the DNA in 40 µL Dilution Buffer prior to use and vortex until solids are completely dissolved and spin down shortly.
Dissolve the crosslinker (brown cap) by adding 100 µL ddH2O and vortex until solids are completely dissolved and spin down shortly. IMPORTANT: Always use fresh compounds.
Add 10 µL of the freshly prepared linker solution to one DNA aliquot. Discard the remaining linker solution from step 3.
Vortex the reactants for 10 sec, spin down and incubate them for 20 minutes at room temperature.
IMPORTANT: Do not exceed incubation time as the reaction yield will decrease.
In the meantime, equilibrate two purification spin columns(red cap) for one coupling reaction:
a. Remove column’s bottom closure and loosen cap (do not remove cap).
b. Place column in a 2.0 mL reaction tube.
c. Centrifuge at 1,500 × g for 1 minute to remove the storage solution.
d. Add 400 µL of Conjugation Buffer on top of column´s resin bed. Centrifuge at 1,500 × g for 1 minute to remove buffer.
e. Repeat step d once, discard buffer from the reaction tube. The Purification spin column should be in a dry state now.
Sample loading
a. Place columns from step 5 in new 1.5 mL reaction tubes.
b. Remove cap of spin column number 1 and apply the sample from step 4 to the top of the resin bed.
c. Centrifuge at 1,500 x g for 2 min to collect the sample (flow- through).
Discard Purification spin column after use.
d. Remove cap of spin column number 2 and apply the sample from step c on top of the resin bed.
e. Centrifuge at 1,500 x g for 2 min to collect the sample (flow- through). Discard Purification spin column after use.
II Protein Conjugation
Add approx. 100 µg (up to 200 µg) of the protein (concentration approx. 0.5 – 50 mg/mL) to the sample from step 6. For optimal conditions use a volume of approx. 50 µL.
EXAMPLE: Adjust protein concentration to 2 mg/mL and use 50 µL for conjugation.
IMPORTANT: Be sure that the storage buffer of the protein does not contain any primary amines, e.g. TRIS buffers, glycine (please see page 4, Important Notes).
Mix the reaction by pipetting up and down and let it react at room temperature for at least 1 hour.
IMPORTANT: Do not vortex. If necessary, the reaction can be carried out at 4 °C with a longer reaction time (e.g. overnight).
III proFIRE® Purification
Please refer to the proFIRE® User Manual.
Perform a purification using the proFIRE®. Please make sure that the sample volume is 160 µL.
- If the volume is less than 160 µL, add Conjugation Buffer.
- If it exceeds 160 µL, please perform two subsequent runs.
Use the Data Viewer Software of the proFIRE® to identify which fractions contains pure conjugate. Example chromatogram:

proFIRE® chromatogram of a protein-DNA conjugate purification.
Used buffers: proFIRE® Buffer A; proFIRE® Buffer B. Column: proFIRE® column. Flow: 1 mL/min. Used program: DNA length 48. Type: 1.
Take the recommended fractions out of the fraction collector.
a. Option 1: Store fractions between 8 °C and -86 °C as desired.
b. Option 2: Proceed with Buffer Exchange and Concentration (see section IV).
IV Optional:Buffer Exchange and Concentration
a. Add 500 µL of the first fraction containing the protein-DNA conjugate from the proFIRE® to the centrifugal filter unit.
Centrifuge at 13,000 x g (up to 14,000 x g) for 10 minutes and discard flow- through.
b. Add the remaining fractions in the same filter unit and repeat the centrifugation step in order to collect all samples in one tube (Please check on page 8: Additional information for the right use of centrifugal filter unit).
c. Add 350 µL of the buffer of choice for buffer exchange and centrifuge at 13,000 x g for 10 minutes. Discard the flow-through again.
d. Add 350 µL of the buffer of choice for buffer exchange and centrifuge at 13,000 x g for 15 minutes. Discard the flow-through again.
e. To recover the protein-DNA conjugate, place the centrifugal filter unit upside down in a new centrifugal collection tube (provided in the kit). Spin for 2 minutes at 1,000 x g to transfer the sample to the tube.
Check protein-DNA conjugate concentration after buffer exchange by using absorbance at 260 nm and the following equation:
*c (protein-DNA conjugate)= A260 nm/(ɛ d)
ɛ = Extinction Coefficient of the DNA
d =** optical path length (usually d = 1 cm, please check photometer manual for further information).
Store between 8 °C and -86 °C as desired.

Additional Information

I Buffer Exchange and Concentration with Centrifugal Filter Units

Nanolever Modification

Take one centrifugal filter unit, add the appropriate volume of buffer in the filter device, and cap it.
Place capped filter device into the centrifuge rotor, aligning the cap strap toward the center of the rotor; counterbalance with a similar device.
Spin the device at 13,000 x g (or 14,000 x g) for the given time.
Remove the flowthrough and repeat the steps 1-3.
Remove the assembled device from the centrifuge and separate the filter device from the microcentrifuge tube.
To recover the conjugate, place the filter device upside down in a clean centrifugal tube, aligning open cap towards the center of the rotor; counterbalance with a similar device. Spin for 2 minutes at 1,000 x g to transfer the sample from the device to the tube.

Useful Order Numbers

TABLE 3 | Order Numbers.

Product name	Order Number
proFIRE® Antibody Oligo Conjugation Kit; sufficient for 3 conjugation series

PF-AB-1
proFIRE® Amine Coupling Kit 1 for proteins (>5 kDa) with thiol-DNA; sufficient for 5 conjugation series| PF-SH-1
proFIRE® Thiol Coupling Kit 1 for proteins (>5 kDa); sufficient for 5 conjugation series| PF-SH-1
Centrifugal filter unit (3 kDa MWCO), 5 pcs.| CF-003-5
Centrifugal filter unit (10 kDa MWCO), 5 pcs.| CF-010-5
proFIRE® column| PF-CC-1
10x proFIRE® Buffer A (50 mL)| PF-BU-A-10
5x proFIRE® Buffer B (50 mL)| PF-BU-B-5
1x Conjugation Buffer (12 mL)| PF-BU-C-1