Technical Note.

Version 0.04
qEV Size Exclusion Column
Beta 1.0 – Specifications and operational guide
Simple Quick Purification of Extracellular Vesicles
Important. Measurements and specifications in this guide relate to beta-test results and should be taken as
indicative of performance. Specifications are subject to change
Features
Izon qEV columns purify biological samples to obtain the extracellular vesicles.
Samples* suitable for qEV use




Blood derived samples, such as serum, plasma
Saliva
Urine
Cell culture media
The advantages gained using an Izon qEV
column include†;
 ~20 minute separation time.
 Greatly reduced risk of protein complex
formation and vesicle aggregation (as can
occur in ultracentrifugation and commercial
exosome precipitation methods).
 Buffers with physiological osmolarity and
viscosity can be used.
 A gentle, rapid method for maximizing
recovery of biological function
 Vesicle recovery is expected to be 50% or
greater
 Protein Removal Ratio > 1000 ‡
 HDL purification > 8 fold
*
Note some ‘raw’ samples cannot be directly run on qEV without preparation steps, such as two or more stage of
centrifugation to prepare the sample (e.g. blood, saliva). Contact [email protected] for assistance with protocols
†
A.N Boing et-al; “Single-step isolation of extracellular vesicles by size-exclusion chromatography”, Journal of
Extracellular Vesicles 2014, 3: 23430 - http://dx.doi.org/10.3402/jev.v3.23430
‡
This reflects the ratio of protein in sample to that eluted in vesicle peak fractions, based on independent testing
of 7 columns, can be as high as 11,000x.
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Specifications
Sample volume
Void volume
Flow-rate
Separation size
Buffer
Bed volume
Largest size passable
Filters (frit) pore size
pH stability working range
pH stability Cleaning-in-Place (CIP)
Shelf life
Life after first use
less than 1ml, 500µl optimal for maximum protein purity
3.0ml ±0.25ml
Typically 0.8 to 1.2 ml/min
70nm nominal
PBS buffer (anti-bacterial <0.05% sodium azide)
10ml
1µm
20µm
3 - 13
2 – 14
>6 months (if stored correctly).
Dependent on use and storage.
Operation
Safety precautions
Always use appropriate personal protection devices such as lab coats, gloves and safety glasses when
handling qEV columns.



The column anti-bacterial solution contain 0.05% w/v sodium azide. Sodium azide in larger
quantities is toxic so direct contact with skin or eyes should be avoided.
Waste buffer should be disposed of in a safe manner. Sodium azide accumulation over time in
copper pipes can result in explosion.
Biological samples can be hazardous, consult you laboratory safety officer for information on
safe handling of your sample when using the qEV column
Storage
Store the column at +4 to +8 °C in the presence of a bacteriostatic agent, e.g. 20%
ethanol, or < 0.05% w/v sodium azide.
Preparation for use



Place the column in a holder and level it (make sure the column is vertical).
Leave the bottom luer-slip cap in place.
Remove the top-cap carefully and slowly. Pinch it tightly so as air can enter
the top of the column as it is being removed and reduce vacuum on the gel.
Column equilibration


Remove the lower luer-slip cap.
Rinse the column with at least 10 ml PBS buffer. Note the
time for 5ml of buffer to flow through in your lab book – this
is useful for detecting when to clean the column.
Sample Application
1.
2.
3.
4.
 Do not allow the column to run dry.
Top filter (frit) must stay wet. If run
dry the column may not function
correctly
 Use only freshly made filtered
(0.2µm) buffer to avoid introducing
particulate contamination
With the bottom luer-slip cap on, pipette out the buffer above the
top filter.
Pipette the sample in.
When ready to begin collecting fractions remove the bottom luer cap.
Immediately start collecting 0.5 ml fractions;
o The first six fractions (3.0 ml) is the void volume and does not contain vesicles so is
usually not analysed.
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o
5.
6.
It is recommended to collect the void volume in one collection tube to save time and
avoid measurement errors of 6 individual tubes.
Add more buffer as the last of the sample just enters (becomes level with) the column top-filter, but add
no more than 2 ml above the top filter.
o Waiting till the last of the sample just enters the top-filter avoids unintentional
dilution of your sample.
Immediately after the void volume collect the vesicle fraction of 1ml. For minimizing protein
contamination collect first 0.5ml only.
For some samples the fraction that the vesicle elution peaks in may be earlier or later
than specified. For the best results collect and measure from 3ml to 4.5ml in 0.5ml
fractions to determine the fraction the vesicle elution-peak, pool fractions as
necessary (noting the dilution effect of pooling multiple fractions).
7.
After collection of the vesicle fractions, flush the column with at least 10ml of buffer and store as
indicated above. Note the time for 5ml of buffer to flow through in your lab book – this is useful for
detecting when to clean the column. A change in flow rate can suggest a blocked or dirty column.
Re-use
How to detect when the column is compromised and needs cleaning;
 Flow rate begins to slow over original flow rate. It is always recommended to measure the rate for the
initial flush (and/or void volume) and note these in your lab book.
 Recovery rate drops significantly. It is difficult to measure the recovery rate accurately as the before
concentration of most biological samples will not be possible to measure easily. It can be inferred when
the expected vesicles recovered reduce with subsequent operation. If this is the case, run Izon CPC100
calibration particles through your column (in PBS buffer), do at least 2000 counts before and after, and
the recovery rate should be within >90% ± 20%
 A colour change from normal at the top of the column
 A space between the top-filter and the gel surface
Regeneration
Regeneration is normally performed by washing with 2–3 column volumes of buffer, followed by reequilibration in the new buffer (if changing conditions).
In some applications, substances such as denatured proteins or lipids do not elute in the regeneration
procedure. These can be removed using the cleaning procedure described below.
Cleaning
Remove precipitated proteins, non-specifically bound proteins and lipoproteins by washing the column with
one column volume of 0.5 M NaOH, then flush the column with at least 3-5 column volumes of PBS and check
the pH of the elution media with litmus paper.
Remove strongly non-specifically bound proteins, lipoproteins and lipids by washing the column with two
column volumes of a non-ionic detergent solution, e.g. 0.1% Triton TM X-100, followed by at least 2–3 column
volumes of elution buffer.
Sanitization
Sanitization reduces microbial contamination of the gel to a minimum. Wash the column with one column
volume of 0.5 M NaOH. Re-equilibrate the column with 3–5 column volumes of sterile buffer and check the pH
of the elution media with litmus paper.
Notes

Degassed buffers will help eliminate air bubbles forming in the gel bed.
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

It is recommended to use a buffer with an ionic strength of 0.15 M or greater to avoid any unwanted
ionic interactions between the solute molecule and the matrix.
To avoid clogging of column filters, it is recommended to filter or low-speed-centrifuge the biological
sample to remove large particulate matter.
Performance Metrics
Peak Elution of Vesicles



The fractions that elution of vesicle peaks in, occurs at 3.5ml ± 0.25ml at loading of 500µl.
Recovery rate in the peak fractions is 75% ± 30%.
For 500µl fractions, the peak fractions are typically fractions 7 and 8. If higher purification is desired,
collect only fraction 7.
Monitoring the A280nm gives indicative protein elution profiles of a qEV column. Measurement of protein in
fractions by using a Bradford assay gives an accurate measurement of the level of protein
qEv: particle and protein elution
10
1.510 1 0
8
1.010 1 0
6
4
5.010 0 9
2
0
Absorbance280
280nm
Absorbance
nm
Particle
ParticleConcentration
concentration
Absorbance 280 nm
Particle Concentration / ml
12
Absorbance 280nm
Particle concentration/ml
2.010 1 0
The enrichment for exosomes and
micro-vesicles is clear, they elute
predominantly in fractions 7, 8, & 9
and the serum protein is retarded
eluting predominantly from F11-F30.
0
0
10
20
30
40
The graph to the left shows the elution
of serum of EV/micro-vesicles
measured by qNano and absorbance
280 nm of each fraction indicating the
relative protein levels of each fraction.
50
no ml)
FractionFraction
number (0.5
Sample Loading Volume
For the qEV loaded with 100µl of serum the relevant fractions can be assayed neat. If the column is loaded
with a greater volume it is recommended to dilute according to Table 1 below, i.e. for 500µl loading on the
qEV column, make a 1:5 dilution i.e. take 100µl of the fraction and add 400µl of electrolyte.
Dilution
200 µl
1:1
300 µl
1:3
400 µl
1:4
500 µl
1:5
nil
Higher loadings result in a lower level of
purity in the microvesicle fractions. The
figure on the right shows the
fractionation of serum from 100 µl, 500
µl, 1000 µl and 2000 µl. Loadings of
1000 µl and 200 µl serum results in
vesicle fractions that are contaminated
with higher amounts of protein. The
delay in elution of exosomes in the 2000
µl loading is apparent.
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When the qEV columns are used according to this protocol and the
fraction volumes are accurate, the exosomes/microvesicles elute
consistently in fractions F7, F8, F9 with low levels in F10 and F11.
5.010 1 0
4.510 1 0
Particle Concentration / ml
Particle concentration/ml
Volume of serum
loaded on qEV
100 µl
4.010 1 0
3.510 1 0
3.010 1 0
100ul serum
500ul serum
1000ul serum
2000ul serum
2.510 1 0
2.010 1 0
1.510 1 0
1.010 1 0
5.010 0 9
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Fraction
(0.5ml)
Fraction
number
(0.5 ml)
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Optimal recommended loadings for purity on the qEV is 100µl and 500µl, which gives consistent vesicles
eluting in fractions 7, 8, 9. The graph below shows qEV elution profiles for these two loadings (using serum).
F10 & F11 usually contain higher protein and low levels of exosomes and these fraction are not recommended
to be used.
In this example, the total combined particle
concentration in F7-F10 for the 100µl loading
was 7.5 x 109/ml. The total combined
concentration of particles from F7-F10 for the
500µl loading was 34 x 109/ml. The recovery
with the 500µl loading was therefore 91%
when compared to the yield for the 100µl
loading§.
1.810 1 0
Particle Concentration / ml
Particle concentration/ml
1.610 1 0
1.410 1 0
1.210 1 0
100
µl serum
100ul
serum
500ul
serum
500
µl serum
1.010 1 0
8.010 0 9
6.010 0 9
Loss occurs with higher loading as the sample
elution is delayed and some particles elute in
fraction 11. This fraction was not analysed
because of the high levels of contaminating
protein expected in this fraction.
4.010 0 9
2.010 0 9
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Fraction (0.5ml)
Fraction number (0.5 ml)
Purification
The following figure shows the indicative improvement in the ratio of vesicles to protein in each fraction, that is;
𝑆𝑡𝑎𝑟𝑡 𝑉𝑒𝑠𝑖𝑐𝑙𝑒𝑠 𝑝𝑒𝑟 𝑚𝑙
⁄𝑐𝑜𝑛𝑐. 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑢𝑔/𝑚𝑙
𝑃𝑢𝑟𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛 =
𝐶𝑜𝑙𝑙𝑒𝑐𝑡𝑒𝑑 𝑉𝑒𝑠𝑖𝑐𝑙𝑒𝑠 𝑝𝑒𝑟 𝑚𝑙
⁄𝑐𝑜𝑙𝑙𝑒𝑐𝑡𝑒𝑑 𝑐𝑜𝑛𝑐. 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑢𝑔/𝑚𝑙
Purification vesicles vs protein
12000.0
Column A
10000.0
Column B
8000.0
Column C
6000.0
Column D
4000.0
Column E
Column F
2000.0
Column G
0.0
fraction 7
fraction 8
fraction 9
fraction 10 fraction 11
Protocols for Preparation from Common EV sources
Biological Samples vary considerably and it is impossible to provide a comprehensive list of protocol
specific to each sample; if you are unsure of what to do to prepare your sample, please contact
[email protected] for assistance.
§
Note, the 100ul loading is 5 times diluted over the 500ul loading.
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Glossary
Chromatography
A method, used primarily for separation of the components of a sample. The
components are distributed between two phases, one is stationary while the other
one is mobile. The stationary phase is either a solid, a solid supported liquid, or a gel.
The stationary phase may be packed in a column, spread as a layer or distributed as a
film. The mobile phase may be gaseous or liquid.
Column volume
Volume of packed material and void volume (can be referred to as the bed volume).
Fraction
Indicates a particular volume collected from column, specified numerically for a given
size. That is to say, fraction 7 of 0.5 ml fractions refers to the 0.5ml volume collected
after 3.0ml and up to 3.5ml
Degassing
Degassing involves subjecting a solution to vacuum to "boil" off excess dissolved air
e.g. applying a vacuum to a flask.
Flow rate
The volumetric flow in ml/min of the carrier liquid
Agarose
High molecular weight polysaccharide used as a separation medium in biochromatography. It is used in bead form, often in gel-filtration chromatography, with
aqueous mobile phases.
Void volume
The total volume of mobile phase in the column; the remainder of the column is taken
up by packed gel material. It denotes the excluded volume in SEC.
Vesicle fraction
The fraction that the vesicles appear in.
Recovery rate
The percentage of vesicles that come out of the column compared with what went in.
Enquire at [email protected] and ask how we can improve the quality of your particle analysis
research.
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