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ISSUE ACTIONS THAT MAY BE NEEDED
Stable baseline current, but
very few particles — Check the dilution factor (use ~ 1:50 for training particles
and ~ 1:1000 for CPC particles ~1:1000). qEV fractions
may be very dilute and may need concentrating.
— Pore is partially blocked by a bubble or an aggregate.
Un-block pores by gently replacing fluid in lower well
and flushing the pore with filtered deionised water at
47 mm stretch as shown on the previous page.
— If working with a calibration standard above 400
nm, sonicate before use. However, please note that
sonication and vortexing can damage biological
samples - only mix with pipettes or by gently inverting/
swirling samples by hand.
— Make up another CPC dilution with 0.22 µm filter-
sterilised measurement electrolyte. All reagents
should be filter-sterilised daily before use.
— Make sure you have coated the pore if working
with biological samples, as non-specific binding of
biological macromolecules to the aperture surface can
cause partial blockages and instability.
— Increase the sample concentration. Use low pressures
until you have a stable system, otherwise you risk
blocking the pore.
— Pore is not the predicted size, try a different
calibration standard or pore.
Sample is blocking the pore
(unstable baseline current) — Run highly-polydisperse samples (e.g. EVs) on two
different sized pores. First, run the sample (with an
appropriate calibration) unfiltered through an NP250
or NP300, and then filter the sample through a 0.22
µm filter and run with a suitable calibration on an
NP100 pore. Once calibrated, the results from both
pores can be displayed on the same plot.
— If only interested in exosomes from polydisperse EV
samples, filter the sample through a 0.22 µm filter
and run with a suitable calibration on a NP100 pore.
— All biological samples should be run through a qEV
SEC column before TRPS analysis, to exclude proteins
that are likely to interact with and block pores.
— Pore is partially blocked by a bubble or an aggregate.
Unblock pores by gently replacing fluid in lower well
and flushing the pore with filtered deionised water at
47 mm stretch as shown on the previous page.
Running calibration standards or biological sample particles
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