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How does the Fluidity One compare to UV-Vis spectrophotometers in testing the concentration of proteins?

How does MDS compare to UV vis

In the quantitation of protein samples, accuracy and range are key. Although UV-vis spectrophotometers such as the NanoDrop allow rapid testing of small volumes, new technologies allow measurements of even lower concentrations.

Microfluidic diffusional sizing, as employed on the Fluidity One instrument, allows concentration determination down to far lower levels with the same ease and speed of use.

What concentration range does each method cover?

The NanoDrop UV-vis spectrophotometer is able to measure from 60 to 820,000 µg/mL.

The Fluidity One can reach 6x lower, with a range of 10 to 500 µg/mL.

How does the workflow compare on each method?

In both instruments a very small (µL) volume of sample is pipetted, then analysis is performed in minutes. Both instruments also offer a touchscreen interface, and the ability to download results in a variety of formats.

When setting up the test on a NanoDrop, you are asked to enter the unique extinction coefficient (ɛ) for your protein. If this is unknown you can select from some pre-defined options, but this does lower the accuracy of the results. Results are obtained in ~5 seconds.

To begin a test on the Fluidity One you must select the size range (from 3 pre-set options, essentially small / medium / large). If the wrong option is chosen, the instrument will recommend another run at a smaller or larger size setting. Results are obtained in ~8 minutes.

How does each method work?

Spectrophotometers like the NanoDrop operate by measuring the absorbance of the sample at 280 nm, which is mostly due to the absorbance of aromatic moieties in tryptophan and tyrosine residues. For proteins with low numbers of these, other methods on the instrument can be selected, although the accuracy of quantitation in the absence of tryptophan residues in particular is low.

The Fluidity One makes use of an optimized formulation of the fluorogenic dye o-phthaldialdehyde (OPA), which reacts with primary amines (for example the N-terminus and lysine side chains) in the presence of a thiol to form a UV-fluorescent adduct. As only the reaction products are active, background fluorescence is low and the sensitivity of protein or peptide detection is high. The fluorescence measurement is performed on the sample, and an on-board calibration function converts the signal obtained directly to concentration. For the few proteins where on-board calibration is not appropriate, users can provide protein-specific calibration by performing a single calibration run.

How does the accuracy of protein concentration results compare?

In NanoDrop results, the accuracy varies greatly dependent on whether the extinction coefficient was known and used or not. As such any results generated with one of the pre-set extinction coefficient values are considered much lower accuracy.

On the Fluidity One concentration results have a <10% accuracy and precision (after calibration where required).

Are there any limitations on either method?

For standard protein concentration at A280 on a NanoDrop, buffers which absorb in this range will affect the results.

On the Fluidity One, samples must not contain free amines – including no Tris buffers.

Summary of UV-Vis absorbance testing vs Fluidity One

Feature

UV-Vis

(Based on NanoDrop One model)

Fluidity One

Measurement method for protein quantification

 

Absorbance at 280 nm due to aromatic chains in Tryptophan and Tyrosine. Microfluidic Diffusional Sizing MDS

Sample volume required 

2 µL

5 µL

Sample knowledge required

 

The unique extinction coefficient for your specific protein improves accuracy.

If this is unknown select from standard “sample type” values.

Estimation of size (must select from S, M, L) – though the instrument will tell you if wrong setting used and recommend larger/smaller

Measurement range

 

On pedestal with A280 method testing BSA;

 

60 – 820,000 µg/mL

=

0.06 – 820 mg/mL

Testing BSA;

  

10 – 500 µg/mL

=

0.01 – 0.5 mg/mL

Measurement time 

5 seconds

~ 6 minutes

Results obtained

 

Protein concentration

(mg/mL or µg/mL selectable)

 

-

 

 

 

Purity Ratio

 

Absorbance at 280 nm

Protein concentration

 

 

 Protein size

(nm – can convert to kDa with online tool)

 

-

 

-

Result storage

 

 

Date/time stamp

 

Free type sample name

 

-

Date/time stamp

 

Free type sample name

 

Add tags with free type values

 

Results export

 

 

 

USB stick / cable

 

.csv

.tsv

.sql (can only be opened in NanoDrop One viewer software)

.nanodrop (Thermo Fisher cloud format)

USB stick / cable

 

.csv

.xls

Different test options available?

As well as standard A280, various options for different protein/peptide types;

 

A205 – peptides, or proteins with high Trp/Tyr

 

Pierce 660 – Detergent compatible. Needs std curve.

 

Bradford – Coomassie blue shift. Needs std curve.

 

BCA – for dilute samples / surfactants. Needs std curve.

 

Lowry – requires std curve.

One test method available.

Reagents required?

 

None

Water and labelling dye – contained in a cartridge and automatically administered so no handling required.

Consumables required? 

None

Disposable chips

User calibration required?

 

 

 

None

 

(Liquid verification kit is available for verification not calibration)

None

Calibration is possible to improve precision if required.

Can pre-labelled proteins be measured?

Yes

No

Limitations

  

 

Cannot report protein size

 

Requires knowledge of extinction coefficient, otherwise low accuracy

No primary amines (inc. no Tris buffers)

Is high throughput testing available?

 

Yes – on one model (NanoDrop 8000) 96-well plates can be tested

No

Can purified and unpurified samples be tested?

 

Purified – A280 or A205 methods

 

Unpurified – select from other methods

Purified – yes

 

Unpurified – no (on future instruments this will be possible)

As both methods present their own strengths and limitations, selecting which method to use will ultimately depend on your specific needs.

For faster, higher concentration, but lower accuracy testing the NanoDrop offers a simple workflow with basic functionality.

For greater accuracy and to test down to lower limits, the Fluidity One caters to users who want a more in-depth analysis.

 

To find out more about the Fluidity One click here, or if you have any questions about protein concentration testing, just contact us here.