reagent kit for trps analysis -...
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REAGENT KIT TECHNICAL NOTE WWW.IZON.COM
1 March 2018
Reagent Kit for TRPS Analysis
Specifications and Operation Guide
Total Measurement Solution
Izon Science's Reagent Kit is designed as part of a total measurement solution for qEV and TRPS
analysis .
qEV Size Exclusion Columns (SEC) isolate extracellular vesicles (EVs) from bio-fluids providing
high-purity vesicles.
Reagent kits provide the components and instructions for the preparation of Nanopore coating
solution and contaminant-free reagents with matched pH and conductivity to ensure reliable
and repeatable measurements using Izon’s qNano.
Izon Control Suite Software (CSS) provides assistants that aid in experimental planning,
capturing the required particle ranges and performing measurements with the attention to
detail necessary at the nanoscale.
Figure 1: The Izon Total Measurement Solution ensures rapid and reliable measurements:
The components of the system combine to produce high quality results.
Reagent Kit
Enhance the reliability and reproducibility of
measurements
Biological particle isolation using qEV
SEC columns
Rapid & effective purification
TOTAL
MEASUREMENT
SOLUTION
qNano with Izon CSS
Repeatable experiment planning and execution.
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Applicable for TRPS analysis of all samples and highly beneficial for biologicals1
For example EVs derived from the following: Blood-derived samples (serum, plasma)
Saliva
Urine
Cell culture media
Cerebrospinal fluid
Advantages of using Izon Science's Reagent Kits
Reliable and consistent results; measurements on different Nanopores are comparable and
accurate
Stable operation, reduces non-specific binding to Nanopores
Contaminant-free solutions
Higher throughput
Safety Data Sheets (SDS)
Safety data sheets for Reagent Kit components can be viewed and downloaded:
http://www.izon.com/products/sds/
Safety Precautions
Always use appropriate personal protection such as gloves, lab coats, and safety glasses when
handling reagents or qEV columns.
There is a trace amount of sodium azide in the wetting solution only. 0.05% w/v
sodium azide is used as an anti-bacterial agent in the wetting solution
concentrate only. Sodium azide in higher concentrations is toxic. Direct contact
with skin or eyes should be avoided.
Waste reagents should be disposed of in a safe manner.
Biological samples can be hazardous; consult your laboratory safety officer for
information on safe handling of your sample when using a qEV column or qNano.
Storage 1Contact Izon Support for assistance with protocols.
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Store the Wetting Solution Concentrate and prepared solutions at +4 to +8 °C. All other Reagent
Kit components should be stored at room temperature. Avoid leaving prepared reagent bottles
open - always replace lids after use and return to the fridge as soon as possible. Observe carefully
the recommended use time of prepared reagents (1 week max) and the expiry date of the wetting
solution.
Operating Instructions in Izon CSS
Standard instructions and measurement protocols for using reagent kits are contained within the
CSS measurement assistants. Contact Izon Support if you need to upgrade or require further
information.
Best Measurement Practices
Careful preparation of reagents and samples for the qNano is critical. Key considerations to get
accurate and reproducible results are:
Use contaminant-free tubes and clean glassware
Wear gloves to avoid contaminating reagent kit components
Dilution errors are a common form of inaccuracy; use calibrated pipettes for dilution
Use a new un-filtered pipette tip each time a reagent is used. This avoids contamination and
ensures the longest life of the reagents
Filter reagents daily using the 0.22 m filters provided, to remove unwanted large
contaminants
Mix samples gently to avoid introduction of bubbles that can interfere with qNano
measurements
Use Izon qEV separation columns to isolate particulates from biofluids, for example
exosomes, microvesicles, viruses etc. to ensure quality TRPS data on biological particles of
interest
Reagent Kit Components
The reagents are tailored to each stage of the measurement. They are designed to control the pH
and electrolyte concentration in order to ensure that measurements on the TRPS system are stable
and physiological. Mixed pH or electrolyte levels can cause instability in measurements. The kit
contains enough components to make four batches of reagents. The reagents included in the kit
are as follows:
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Coating Solution (4 x 20mL, provided as a powder to be prepared)
A critical part of the Reagent Kit is to minimise the non-specific binding (NSB) of biological
molecules/particles to the Nanopore that can interfere with EV measurements. In TRPS, NSB can
interfere with measurements in two ways – firstly, contaminating molecules can bind to the pore,
altering the membrane properties, and secondly, EVs themselves can bind to the membrane,
causing it to block and preventing further measurements.
qEV purification greatly reduces the problems caused by NSB; however, pre-treatment of the
Nanopore is necessary to prevent NSB of low-level contaminants that may remain after
purification.
Without Coating Nanopore With Izon Coating Solution
Figure 2: Contact with biological fluids alters the properties of the Nanopore: Exposure of the pore to
serum proteins shows that the IV curve (a measure of the pore performance) is significantly altered. When the
Nanopore is pre-coated its performance is unaltered leading to repeatable calibrated measurements.
The coating solution is used to pre-coat the Nanopore prior to sample analysis. It is also added to
the measurement electrolyte at a lower concentration, to maintain a low volume of coating reagent
throughout the measurement process.
Store the powder well sealed at room temperature.
Shelf life: 1 week when made up as a solution, if stored at 4-8°C
Glassware for mixing PBS must be highly clean and devoid of particulates, washed with de-ionized (DI) water. Use high-quality plasticware for reagent mixing and sample preparation e.g. 1.5 mL, 15 mL, 50 mL plastic tubes, we recommend the use of Axygen
Scientific tubes.
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PBS tablets (4 tablets)
The supplied PBS tablets are used to prepare stock PBS solutions solely for TRPS and qEV columns.
Reagent quality is highly important for TRPS. Do not use general purpose lab PBS. The stock PBS
is used to prepare the measurement electrolyte which is used extensively through the
measurement process. The measurement electrolyte is also used as the diluent for samples and
calibration particles.
If using a buffer other than the PBS buffer provided substitute the alternate buffer where PBS stock
solution is mentioned in the steps below.
Store at room temperature.
Shelf life: 1 week when made up as a solution, if stored at 4-8°C
Wetting Solution Concentrate (4 mL)
Wetting solution concentrate is added to the measurement stock solution as a surfactant.
Store the at 4-8°C
Shelf life: 6 months once opened, if stored at 4-8°C
Syringe Filter Unit, 0.22 µm (20 pieces) and 0.45µm (4 pieces)
Single use 0.45 µm filters are provided to filter the coating solution when it is first prepared (Step
2 below).
Single use 0.22 µm syringe filters are provided to filter flushing water, refilter an aliquot of the
coating solution and measurement electrolyte (in this sequence) before use each day (Step 4
below). A critical part of the measurement of nanoparticles is high-purity reagents. It is necessary
to filter all solutions to remove any large particles which will block the Nanopore.
Store well sealed at room temperature.
Solution Preparation
All unused stock solutions must be stored in the fridge and discard after one week. Follow the
steps below to prepare the measurement solutions.
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Particle Preparation
Calibration Particles
All calibration particles must be diluted from concentrated stocks immediately before use, using
Measurement Electrolyte. Each Nanopore size has an associated target particle concentration.
Consult the Nanopore Reference Guide in the qNano User Manual for further details.
•Stock PBS Solution must be prepared weekly and unused solution should be discarded after one week.
•To make the Stock Solution: •Completely dissolve one PBS tablet in 200 mL of DI water using clean
glassware. •Add 0.6 mL of Wetting Solution Concentrate to the resultant solution
and mix evenly. •Seal container and label as Stock PBS Solution. •Store in the fridge and discard after 1 week. •Allow solutions to warm up to room temperature before use.
1. Prepare Stock PBS Solution
•Add 10 mL of Stock PBS Solution to a 50 mL graduated plastic tube. •Accurately weigh out 2.0g of the coating solution powder and add this to
the 50mL tube. •Add Stock PBS Solution to bring the volume to 20 mL. Apply the lid. •Place the 50 mL tube and contents into a 50 °C water bath or beaker
containing warm water.
•Once warm mix by swirling at regular intervals until the powder is totally dissoved.
•When the solution is clear, check the volume and top up to 20 mL with Stock PBS Solution if necessary.
•Filter through a 0.45 m filter into a clean tube labelled Coating Solution. •Store in the fridge and discard after 1 week.
2. Prepare Coating Solution
•Measurement Electrolyte should be made fresh daily. 15 mL of Measurement Electrolyte should suffice for a day's work.
•To make the Measurement Electrolyte : •Vortex the Coating Solution in short bursts for 20 seconds. •Using a new pipette tip, pipette 3 mL of Coating Solution into a clean
15mL tube. •Add 12 mL of Stock PBS Solution to the tube. •Vortex the resultant solution in short bursts for 20 seconds.
3. Prepare
Measurement Electrolyte
•Filter 15 mL of DI water into a clean 15 mL tube using a 0.22 m filter,
•Using the same 0.22 m filter, filter 0.5 mL of Coating Solution into a clean 1.5 mL tube labelled Coating Solution. (This aliquot will be used during the system set up and prevents contamination of the bulk solution).
•Finally using the same 0.22 m filter, filter the Measurement Electrolyte from step 3 into a clean 15 mL tube. Discard the filter.
•Discard these solutions after 1 day.
4. Filter
Solutions
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Sample Particles
Correct sample preparation will provide faster and more accurate results. For the best results Izon
Science recommends the use of qEV separation columns for biological samples. Each sample may
require slightly different preparation.
Follow the protocol outlined in the Izon qEV Technical Note to isolate the EVs from the raw
sample. When using qEV separation columns, reagent quality is critical. Izon recommends
the use of Sigma-Aldrich PBS tablets (Cat# P4417) with high-purity water.
Dilute the qEV fraction(s) in Measurement Electrolyte. The dilution should be optimised
to achieve a particle rate at the highest operating pressure of 1000-2000/min to avoid pore
blockage.
If an initial approximate concentration of the sample is unknown, a series of samples may
be prepared at different dilutions, e.g. 1:100, 1:10, 1:5. For EV samples from qEV purified
plasma we recommend 1:5 to 1:10 initially.
Highly Polydispersed samples: If the sample contains very large contaminants that create
problems for TRPS analysis, consider filtering the sample. Izon recommends Millipore spin
filters (Ultrafree –MC centrifugal filter units for exosome samples). Note as these remove
larger particulates the data will be biased to some degree.
Nanopore Setup Overview
Users should complete an Izon Training Programme before attempting to measure their own
samples. The chart below shows an overview of Nanopore setup:
Prepare Measurement
Electrolyte and Sample Particles
Prepare the Fluid Cell
Fit and calibrate Nanopore
Wetting Protocol1 Coating Protocol2 Equilibrate
Measurement Electrolyte3
Insert Calibration Particles
Optimise stretch, voltage, and
pressure
Sample and calibration Recordings
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Clicking is only recommended during Nanopore wetting.
Step Procedure
Wetting Protocol1
1. Load 35 µL of Measurement Electrolyte in the
upper fluid well ONLY.
2. Apply maximum pressure for 2 minutes.
3. Click the shielding lid 5 to 10 times to
mechanically assist wetting the Nanopore.*
4. Add 75 L of Measurement Electrolyte to the
lower fluid well and verify that the Nanopore is
wetted by applying an appropriate voltage and
checking for a stable open Nanopore current.
Coating Protocol2
1. Load filtered Coating Solution in the upper and
lower fluid well.
2. Apply maximum pressure for 10 minutes.
3. Apply maximum vacuum for 10 minutes.
Equilibrate Measurement
Electrolyte3
1. Flush the coating solution out of the upper and
lower fluid wells two to three times with
Measurement Electrolyte.
2. Apply maximum pressure for 10 minutes.