qEV columns utilise size exclusion chromatography (SEC) to separate a sample's components based off size. This allows for the purified collection of extracellular vesicles (EVs).
The Automatic Fraction Collector (AFC) is designed to process samples going through qEV columns (excluding the qEV100 – for that, you’ll need a different sort of automation assistance – enquire within!) in a hands-off, yet precise manner.
Through automation, the AFC brings a multitude of advantages when compared to manual collection. These include:
- Increased uniformity of collected volumes – collect the same volume every time
- More free time in the lab – get other samples organised while you let the AFC run
- Elimination of manual collection inaccuracies
- Scalability through tailored PCV collection – customise parameters to best suit your preferences
How does the AFC work?
The AFC operates by measuring the volume of eluate as it exits the qEV column, with the eluate weighed in the collection tubes. As a sample runs through the column, the components are separated by size due to principles of size exclusion chromatography (SEC). The first volume to elute is the buffer volume (BV), which typically is discarded as waste and contains very few, if any, EVs. Following the buffer volume, the purified collection volume (PCV) elutes, containing EVs that quickly flow through the column’s resin, largely bypassing pores in the resin. Last to elute are smaller, contaminating proteins and other small molecules that get trapped in the resin pores on their way through the column.
The AFC comes with a pre-set default BV and default PCV that are specific to the type of qEV column used (recognised via the RFID tag). Both the default BV and the default PCV are determined using human plasma.
Collecting your PCV in fractions (e.g., as 4 x 0.7 mL volumes using the qEV1) allows you to determine your elution profile. Alternatively, for a simpler and streamlined approach, the PCV can be collected as a single volume (as 2.8 mL, in the case of the qEV1).
The value of an elution profile in EV studies
An elution profile is exactly that – a portrait of your sample's eluate characteristics. The sample's elution profile provides you with insights such as when you begin isolating EVs, and when this transitions into the collection of contaminants (such as proteins). Once you have obtained an elution profile (Figure 1), it is strongly recommended you adjust the buffer volume and PCV parameters to best suit your sample type and preferences.
Figure 1. Elution profiles of qEVoriginal columns (20 nm, 35 nm Gen 2, and 70 nm Gen 2) with 0.5 mL of human plasma loaded. Particle concentration was measured using the Exoid, and protein concentration was measured using a bicinchoninic acid (BCA) assay. Particle concentration is expressed as the mean ± standard error, while the percentage of protein recovered is depicted using the mean value. n=3 for each column series. BV = Buffer Volume; Default PCV = Default Purified Collection Volume.
Choosing your PCV
Although qEV columns have been designed to maximise the separation of the EV- and protein-containing peaks, there will be some degree of overlap between the peaks which requires you to make a judgement call on where you draw the line with the PCV: do you collect a little more, at the cost of having a bit more protein in your purified sample? Or, do you reduce your PCV, sacrificing a few EVs but maximise purity? This decision will be personal to you and your situation.
The benefits of single volume collections
Collecting the PCV in distinct, small volumes (as fractions) is not always useful. Once you have a clear understanding of your sample’s elution profile, you may wish to simply collect the PCV in one larger volume up to 5 mL.
A single volume collection by the AFC prevents the need to manually pool your samples, which can lead to sample errors downstream or loss of sample.
Best of all – single volume collection is easy. The AFC's user-friendly interface allows you to adjust the buffer volume, number of fractions, and fraction volume you would like to collect (Figure 2).
So, back to the qEV1 example – instead of collecting 4 x 0.7 mL fractions, you can collect 1 x 2.8 mL volume. All you need to do is adjust the number of fractions (‘’Count’’) to one, change the fraction volume (‘’Volume’’) to the total PCV you are collecting (up to 5 mL), and flip the carousel to accommodate your 5 mL collection tubes. It's really that easy!
Learn more about the Automatic Fraction Collector
