dynamic BIOSENSORS heliX plus Reducing Agent Kit 1 User Manual
- August 27, 2024
- dynamic BIOSENSORS
Table of Contents
dynamic BIOSENSORS heliX plus Reducing Agent Kit 1
Key Features
- Allows for reduction of disulfide bonds using TCEP.
- Compatible with proteins and antibodies (MW > 20 kDa).
- Site specific reduction of antibodies.
- Immediate coupling of reduced molecules is possible (refer to user manual HK-MAL).
- Reduction of multiple ligands can be performed at the same time.
- Includes reagents for two individual reduction reactions.
Product Description
Order Number: RK-PA-1
Table 1. Contents and Storage Information
| Material | Cap | Amount | Storage |
|---|---|---|---|
| TCEP (500 mM) | Red | 2 x 20 µL | -20°C |
| Reducing Buffer [1] | White | 2 x 1.8 mL | -20°C |
| Purification spin column | Red | 4 x | 2-8°C |
| 2.0 mL reaction tubes for purification spin column | 4 x | RT | |
| Centrifugal filter unit (10 kDa MWCO) | 2 x | RT | |
| Centrifugation collection tube | 4 x | RT |
For research use only.
This product has a limited shelf life, please see expiry date on label.
IMPORTANT| Products may be shipped at different temperatures, but storage
should adhere to the guidelines outlined in the Table. The resin slurry in the
purification spin column contains 0.02 % sodium azide.
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Additional Materials Required
Table 2. Additional Materials
| Material | Comments |
|---|---|
| Benchtop microcentrifuge | Required speed range of between 1,000 x g to 13,000 |
x g
Vortex|
1.5 mL reaction tubes|
All necessary solutions and buffers are included in the kit.
3-Step Reduction of a Biomolecule in a Reaction Tube
Please read the entire protocol before starting and perform all steps without interruption.
TIP| This protocol can be performed simultaneously for multiple coupling
reactions
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Buffer Exchange of Protein/Antibody
- Wet the centrifugal filter unit membrane with 100 µL Reducing Buffer.
- Add approx. 200 µg (up to 500 µg) of protein/antibody to the filter unit from step 1. If necessary add Reducing Buffer to the filter until a maximum volume of 450 µL is reached and centrifuge at 13,000 x g (up to 14,000 x g) for 5 min and discard flow-through.
- Add 350 µL of Reducing Buffer and centrifuge at 13,000 x g for 5 min and discard flow-through again.
- Repeat step 3, one more time with a final centrifugation time of 10 min to get a final volume of approx. 80 µL.
- Collect sample either with a 100 µL pipette into a new tube or by placing the filter device upside down in a new centrifugal tube, centrifuge for 2 min at 1000 x g.
Reducing with TCEP
-
Prepare a 2.5 mM TCEP solution in Reducing Buffer by adding 1 µL of 500 mM TCEP into 199 µL Reducing Buffer.
Prepare always directly before use. -
Add Reducing Buffer to the sample vial for a total volume of 98 µL and add 2 µL of 2.5 mM TCEP solution to obtain a final concentration of 50 µM TCEP. Mix the reaction by pipetting up and down.
-
Incubate the solution at 37°C at 400 rpm for 2 h to ensure complete reduction.
Purification and Concentration
- Equilibrate two purification spin columns (red cap) for one TCEP wash out:
a. Remove the column’s bottom seal and loosen cap (do not remove cap).
b. Place the column in a 2.0 mL reaction tube.
c. Centrifuge at 1,500 × g for 1 min to remove the storage solution.
d. Add 400 µL of Reducing Buffer to the column´s resin bed. Centrifuge at 1,500 × g for 1 min to remove buffer.
e. Repeat step d and discard the resulting buffer from the reaction tube. The purification spin column should now be in a dry state.
TCEP wash
a. Place the columns from the previous step in new 1.5 mL reaction tubes.
b. Remove the cap of spin column number 1 and pour delicately the sample
to the top of the resin bed.
c. Centrifuge at 1,500 x g for 2 min to collect the sample (flow-
through). Discard the purification spin column after use.
d. Remove the cap of spin column number 2 and pour the sample from the
previous step to the resin bed.
e. Centrifuge at 1,500 x g for 2 min to collect the sample (flow-
through). Discard the purification spin column after use.
f. Thiol coupling according user manual (HK-MAL-1 or HK-MAL-2) of reduced
sample can be performed.
Additional Information
Buffer Exchange and Concentration with Centrifugal Filter Units
- 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 flow through and repeat 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 | Amount | Order No |
|---|---|---|
| heliX ® Thiol coupling kit 1 | 5 x | HK-MAL-1 |
| heliX ® Thiol coupling kit 2 | 5 x | HK-MAL-2 |
| Centrifugal filter unit (3 kDa MWCO) | 5 pcs. | CF-003-5 |
| Centrifugal filter unit (10 kDa MWCO) | 5 pcs. | CF-010-5 |
| 10x Buffer A [2] | 50 mL (yielding 500 mL) | BU-P-150-10 |
| 5x Buffer B [3] | 50 mL (yielding 250 mL) | BU-P-1000-5 |
Contact
| Dynamic Biosensors GmbH | Dynamic Biosensors, Inc |
|---|---|
| Perchtinger Str. 8/10 | 300 Trade Center, Suite 1400 |
| 81379 Munich | Woburn, MA 01801 |
| Germany | USA |
Order Information order@dynamic-biosensors.com
Technical Support support@dynamic-biosensors.com
- Reducing Buffer : 100 mM Na2HPO4/NaH2PO4, 300 mM NaCl, 5 mM EDTA, pH 7.4
- Buffer A : 50 mM Na2HPO4/NaH2PO4, 150 mM NaCl, pH 7.2
- Buffer B : 50 mM Na2HPO4/NaH2PO4, 1 M NaCl, pH 7.2
CUSTOMER SUPPORT
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Instruments and chips are engineered and manufactured in Germany.
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