Hi, my name is Struan and I’m a Scientist at Izon Science and today we’ll be going through manually running a qEV column.
qEV columns are a quick and effective tool used to remove contaminating proteins from EV samples,
with gen 2 columns removing approximately 99% of protein from biological samples such as human plasma.
The qEV column range can handle sample volumes from as little as 150 µL to 100 mL,
making them a versatile separation method.
Before loading the qEV column with a biological sample, it needs to be prepared accordingly.
Instructions for how to prepare plasma samples can be found in the qEV user manual,
instructions for other sample types,
such as cell culture media and urine,
can be found at support.izon.com.
Overall, running a qEV column requires you to prepare enough of your elution buffer to flush the column,
run your sample and clean the column afterwards,
this is typically 6 to 8 column volumes of buffer.
The buffer should also be freshly degassed to prevent air bubbles forming in the gel bed.
Label a waste vessel,
a buffer volume tube if you are interested in analysing the buffer volume,
and your sample collection tubes.
The sample collection vessels can be one tube if you are interested in a pooled sample or multiple tubes if you are interested in collecting an individually fractionated sample.
Once your elution buffer and sample have been prepared,
and all your tubes are labelled,
you are now ready to start running the column.
For the qEV single,
qEVoriginal or qEV1,
remove the upper cap from your column first,
ensuring the lower cap is still in place.
If you remove the lower cap first you will damage the column when the upper cap is removed.
With the waste tube in place to collect the fluid, remove the lower cap
and the column should start to flow.
The liquid will flow down as far as the upper FRIT and then stop.
For the qEV2 or qEV10,
first wet the reservoir with enough buffer to fill the gap between the FRIT and the tip of the column.
Then remove the top cap from the column and check for any air in the junction.
If there is any air top the connector up with buffer so that there is a small volume of buffer on top of the column connector.
Connect the reservoir to the upper junction firmly.
Remove the bottom cap and check that the column begins to flow,
until the buffer reaches the reservoir FRIT,
at which point it should stop flowing.
If the buffer continues to flow out the bottom of the column after the buffer has finished entering the FRIT of the reservoir,
then there is air in the junction that needs removing.
A guide on how to remove air in the junction can be found at support.izon.com.
qEV columns should be flushed with at least two column volumes of PBS buffer before use.
If you are using a buffer other than PBS, flush with 3 column volumes of your buffer.
Once the column has been flushed,
put your collection vessel in place under the column; this will either be your labelled buffer volume collection vessel,
if you do not wish to collect the buffer volume,
this will be the waste vessel.
Then you can load your sample.
For the qEV original,
we are loading 500 µL of plasma while on the qEV10 we are loading 10 mL of concentrated cell culture media.
Allow the sample to run completely into the FRIT before adding any buffer.
Once the sample has passed into the FRIT,
add enough buffer so that the total volume of your sample and the added buffer is equal to the buffer volume of your column.
For example,
on the qEV original gen 2 we added 0.5 mL of sample and the buffer volume is 2.5 mL,
so add 2 mL of buffer.
Then let it collect in the buffer volume collection vessel.
For the qEV 10 gen 2, we’re adding 12.9 mL of buffer to reach the total buffer volume of 22.9 mL.
Once the buffer volume has been collected, set it aside and load your first sample collection vessel.
Once your collection vessel is loaded,
pipette the collection volume for the fraction you wish to collect into the top of the column,
or the buffer reservoir.
For example,
if you are collecting individual fractions on a qEV original gen 2 optimising for EV purity,
then you would add 0.4 mL of buffer to the column,
then collect two more fractions of 0.4 mL each.
Once you have collected your sample, follow the cleaning instructions for the qEV column as shown in the user manual.
For the qEV original gen 2 this involves loading 8.5 mL of 0.5 M Sodium hydroxide solution,
once this has run through,
flush the column with 17 mL of buffer.
To store your qEV column,
it is recommended to flush with a bacteriostatic buffer,
such as PBS with 0.05% sodium azide,
or 20% ethanol solution.
If you are storing in 20% ethanol solution,
it is recommended that you flush the column with two column volumes of water after cleaning,
then introduce the 20% ethanol solution into the column.
This will help prevent salt precipitating in the column when the ethanol is introduced.
Izon qEV columns are suited to 5 uses if cleaned and stored following the protocols outlined in the user manual.
If you want to further simplify,
scale and standardise the process,
we recommend considering the automatic fraction collector for your sample separation.
If you have any questions or would like to know more, please contact us at support@izon.com..