Tech Note Guide

Table of Contents

Ease of Use ..................................................................................................................................................... 3

Sensitivity ....................................................................................................................................................... 4

Resolution ...................................................................................................................................................... 5

Accuracy ......................................................................................................................................................... 6

Faster Sample Prep ........................................................................................................................................ 7

Measuring Molecular Weight ........................................................................................................................ 8

Observing Protein Aggregation ...................................................................................................................... 9

Characterize Protein Solutions .................................................................................................................... 10

Online Measurements ................................................................................................................................. 11

Measuring the < 1 nm Range ...................................................................................................................... 13

Rules of Thumb ........................................................................................................................................... 14

Technical Note

3

Using rUNt Disposable Cuvettes Unchained Labs, supplies 5μl rUNt disposable sample cuvettes with every pUNk DLS system. Each rUNt is precision molded in black polycarbonate material and cleaned prior to packing. The patent pending design can be used in the pUNk as a direct replacement for traditional quartz non-disposable sample cuvettes. Sample Preparation

No special preparation is required beyond normal procedures recommended for DLS. Filtration is not required, however it is advisable to centrifuge to reduce the possibility of dust contamination affecting the measurement. Filling the rUNt

5μl of sample is simply pipetted directly against the walls of the liquid trap where it is retained by surface tension alone. Making a Measurement

The prepared cuvette is placed into the temperature controlled sample holder of the pUNk system, where it can be analyzed by DLS in seconds. When the measurement is complete, the rUNt may be removed. Sample Recovery

The sample is recovered by simply dipping the pipette tip into the liquid trap and removing the contents. The rUNT may now be disposed. Summary

The rUNT is a ground breaking innovation making very low volume batch DLS measurements easier and more convenient than ever before. Unlike quartz cuvettes, which have to be cleaned, there is no risk of cross-contamination. The rUNT cuvette and the pUNk DLS platform are only available from Unchained Labs.

Technical Note

4

pUNk Sensitivity: Measuring 0.1mg/ml Lysozyme The Unchained Labs pUNk dynamic light scattering system and software provide a quick and accurate measurement of hydrodynamic size of sub-micron particles and molecules. To measure extremely low concentrations of weakly scattering protein molecules requires cleanliness, care and patience, but follow these basic rules and it is perfectly possible to measure just 0.1mg/ml of lysozyme (14kDa monomer) with confidence. Sample Preparation & Measurement

A concentration of 0.1mg/ml lysozyme powder in filtered PBS was prepared and allowed to settle for 1 hour. 12μl of the prepared solution was filtered through a 0.02μm pore size and then pipetted into a scrupulously cleaned, quartz glass cuvette. The prepared sample was placed in the temperature controlled cell holder and measured by the pUNk for a total of 60 seconds.

Results

The resulting correlation function and size distribution reported by the pUNk software indicates a single peak of radius 1.9nm, which is appropriate for lysozyme. Summary

Measuring at the limits of DLS sensitivity requires care, but the pUNk DLS and software are powerful enough to produce reliable results from the most extreme challenges.

Technical Note

5

pUNk Resolution

The Unchained Labs pUNk dynamic light scattering system and software provide a quick, convenient and accurate measurement of hydrodynamic size of sub-micron particles and molecules. Using the unique 5μl rUNt disposable cuvette, this technical note illustrates the capability of discriminating a polydisperse suspension of latex particles.

Sample Preparation & Measurement

A mixture of approximately 100 parts 22nm to 1 part 200nm diameter latex particle size standards was dispersed in a phosphate buffer. 5μl of the prepared suspension was pipetted into a rUNT cuvette and

measured by the pUNk for a total of 50 seconds. Results

The resulting size distribution reported by the pUNk software indicates two clearly separated peaks of 11nm (22nm diameter) and 95nm (190nm diameter). Each peak scatters roughly half the measured light intensity while a tiny third peak is an artifact of the distribution calculation and can safely be ignored. Summary

pUNk software can easily discriminate a polydisperse sample where there is sufficient size differential, however the size groups must be unequally proportioned in favor of the smaller size by mass (number) to produce equal amounts of scattered light.

Technical Note

6

Measurement Accuracy of the rUNt Disposable Cuvette Background: The pUNk dynamic light scattering (DLS) system from Unchained Labs uses the 5μl rUNt disposable sample cuvettes and is also capable of utilizing a wide range of standard, square walled measurement cuvettes. It is important to be sure the accuracy and quality of the measurement results comply with the requirements of ISO22412:2008 the International Standard for dynamic light scattering. In this Technical Note, we show how the results obtained from a rUNt comply with the standard and are the equal of traditional square walled disposable cuvettes but use only a tiny fraction of the sample volume. Sample Preparation: A preparation of 100nm and 200nm diameter NIST traceable certified size standards (Thermo Scientific 3100A & 3200A) was dispensed into 5μl rUNt and 4.5ml square walled disposable cuvettes (Kartell brand #1960). Measurement: The prepared cuvettes were each placed into the temperature controlled sample holder of the pUNk DLS system and allowed to stabilize at 20°C. A series of acquisitions according to ISO22412 were obtained for each cuvette type and the averages calculated.

Cuvette Type Certified Mean Diameter Measured Mean Diameter Pd Index (<0.1) 5µl rUNt 93.1-108.1 99.9 0.017

4.5ml Kartell #1960 (100nm nominal diameter) 98.0 0090 5µl rUNt 192.1-210.1 203.9 0.071

4.5ml Kartell #1960 (200nm nominal diameter) 198.0 0.044 Results: The mean size measured in each cuvette type was found to be within the specified size range according to the ISO standard. The Polydispersity Index values do not exceed the maximum permitted value of 0.100. Summary: The pUNk DLS system is sup- plied with 5μl rUNt disposable cuvettes as standard. The accuracy of the measurement results comply with ISO22412:2008, the International Standard for dynamic light scattering.

Technical Note

7

8X Faster DLS Sample Preparation with the pUNk The pUNk from Unchained Labs is the only dynamic light scattering (DLS) system in the world with patent pending 5μl rUNt disposable sample cuvettes. Other leading DLS systems use traditional, non-disposable quartz glass cuvettes for low volume work, which must to be cleaned between uses. Cleaning is a tiresome chore and introduces the risk of cross-contamination between samples. A study by Unchained Labs illustrates the extent of the time taken between useful DLS measurements when compared to the unique convenience of the rUNt. Traditional Quartz Cuvette

Total Sample Prep Time = 240 seconds

20 separate activities totaling 4 minutes

Cuvette preparation (total = 100 seconds) • Visual inspection through windows (5s) • Pipette filtered water into cuvette (10s) • Replace cap and clean windows (10s) • Insert into sample holder (5s) • Observe scattering intensity to ensure cleanliness (20s) • Remove cuvette (5s) • Use pipette to remove water (15s) • Dry cuvette using dry gas or air duster (30s)

Sample loading (total 20 seconds)

• Visual Inspection (5s) • Pipette sample into cuvette (10s) • Insert cuvette in holder (5s)

Measurement process (sample dependent time)

Sample recovery (15 seconds) • Remove cuvette (5s) • Recover sample (10s)

Cuvette cleaning (total =105 seconds)

• Dispense detergent solution into cuvette (5s) • Use Pasteur pipette to flush cuvette with detergent

solution (10s) • Remove detergent solution (10s) • Use Pasteur pipette to flush cuvette copiously with

running water (30s) • Remove water and dry with air duster (30s) • Replace cuvette cap and clean cuvette windows (10s)

rUNt Disposable Cuvette Total Sample Prep Time = 30 seconds 5 separate activities totaling 0.5 minutes

Cuvette preparation (total = 0 seconds)

Sample loading (15 seconds) • Pipette sample into cuvette (10s) • Insert cuvette in holder (5s)

Measurement process (sample dependent time)

Sample recovery (total = 15 seconds) • Remove cuvette (5s) • Recover sample and dispose of cuvette (10s)

Cuvette cleaning (total = 0 seconds)

Technical Note

8

Measuring Molecular Weight with the pUNk Background: Laser light scattering theory predicts that when a molecule is much smaller in size than the incident laser wavelength, there is very little angle dependence in the measurement and it is therefore possible to accurately measure molecular weight using a single scattering angle. The Molecular Weight Calculator feature of the pUNk software provides this useful facility and is based on the theory given by the Rayleigh equation for molecular weight measurement. Measurement Procedure: A series of concentrations is injected manually through a flow cell placed in the sample holder of the pUNk DLS system. Starting with pure solvent, followed by each increasing concentration the sample scattering intensities are recorded using i-Size software. Results: The time averaged scattering intensities and sample concentrations are entered into the Molecular Weight Calculator together with the solvent refractive index and count rate measured from a toluene reference standard. A Debye plot is produced and the molecular weight, Mw and 2nd Virial Coefficient, A2 are displayed in red.

Summary: The pUNk DLS system can be used to accurately measure molecular weight of proteins in solution. The same DLS system can also produce quick and accurate measurements of molecular size and aggregation state utilizing Unchained Labs unique disposable 5μl rUNt cuvettes.

Technical Note

9

Observing Real Time Protein Aggregation Using the pUNk

Background

Dynamic light scattering (DLS) is widely used to detect the size and distribution of molecules, particles and their aggregates under a variety of solvent conditions. In a recent case study the speed and power of DLS was demonstrated by revealing the spontaneous self-association of a molecule in solution. Dynamic processes can be easily observed in real time by DLS due to the short acquisition times and ‘ensemble’ measurement method. Experiment & Results

In our study, a peptide compound in solution was initially shown to contain a mixture of monomer and aggregates when measured on the pUNk DLS system. To reduce the aggregate content the sample was centrifuged at 10,000 rpm for 10 minutes. An aliquot was re-analyzed to reveal a single peak of narrow size distribution and no aggregate peak (Fig 1). A second measurement was taken four minutes later (Fig 2) and on this occasion a small secondary peak was detected. Subsequent measurements at later time intervals indicated an increasing light scattering signal from the aggregate content (Fig 3) while the monomer peak remained constant in size. The process continued for 45 minutes until the original monomer/ aggregate equilibrium was restored (Fig 4). Conclusion

DLS measurements are performed quickly and non-invasively, allowing dynamic processes to be observed very easily and with great sensitivity.

Technical Note

10

Characterization of High Concentration Protein Solutions with the pUNk Background

Dynamic light scattering (DLS) is widely used to detect the size and distribution of molecules, particles and their aggregates under a variety of solvent conditions. In the field of protein crystallography, the process of increasing the concentration of protein solutions required for crystallization often encourages unwanted aggregation, however some DLS systems are incapable of detecting these aggregates due to excessive light scattering from the concentrated sample. The pUNk DLS system from Unchained Labs incorporates an automatic, wide range optical attenuator, which permits measurement of protein samples at concentrations far in excess of what is possible with other DLS systems. Experiment & Results

Concentrations of 50, 75 & 100mg/ml BSA protein (66kDa MW) was prepared in a phosphate buffer and analyzed using the pUNk software and Unchained Labs’ unique 5μl rUNt sample cuvettes. All samples produced distributions with small secondary aggregate peaks, which is common for very high concentrations but in the case of BSA is not severe enough to inhibit crystal nucleation and growth. At 50mg/ml, the measured size and MW estimate are very close to the true values for BSA. At 75 and 100mg/ml there is a noticeable incremental decrease in reported sizes and corresponding MWs. Conclusion

The raw DLS data was of high quality and the preparations optically transparent so the apparent size change is unlikely to be caused by multiple scattering phenomena and more likely due to molecule-molecule interaction. The pUNk DLS system automatically compensates for highly scattering samples making it possible to measure the size and distribution of proteins at a wide range of concentrations, however at very high concentrations the measured size may differ from the true size due to molecule-molecule interaction.

Technical Note

11

Online Hydrodynamic Size Measurements Using the pUNk

Background

The speed and sensitivity of dynamic light scattering lends itself to online applications such as chromatography of proteins. Size Exclusion Chromatography (SEC) separation systems are routinely used for molecular characterization and purification. The pUNk DLS system from Unchained Labs can be fitted with a low volume flow cell and simply connected to any chromatography system to provide instant online measurement of hydrodynamic radius (Rh) and scattering intensity as well as for detection of aggregates.

SEC systems use UV detection to determine the elution times of each size component and there- fore provide an indication of the size distribution and aggregate content. The addition of (static) light scattering provides molecular weight meas- urement capability, however this requires calibra- tion with standards. DLS does not require cali- bration to measure Rh. In addition to directly measuring Rh and intensity, the molecular weights of globular species may be reliably estimated using the proprietary algorithm built into the pUNk DLS software.

Method

The pUNk DLS system flow cell tubing is connected inline after the separation column or UV detector before going to fraction collection or waste. As soon as the sample is injected or once the UV peak begins to appear, DLS acquisitions are initiated until all peaks pass through. The collected DLS and UV data is exported directly to a spreadsheet application for further analysis and correction for inter-detector volume if desired.

Typical Samples: 25kDa@5mg/ml 100kDa@1mg/ml Injection Volume : 50uL

Typical Column : 2.4 ml GE Superdex 200 Flow rate : 500uL/min SEC Temp : 15 C

DLS System: Acq. Time : 5sec # of repeats : 180 (15 mins) Laser output : 100% DLS Cell Temp : 15 C

Technical Note

12

Compact size of pUNk system makes hardware placement within cold cabinet possible

Results Directly measuring Rh and scattering intensity every few seconds provides a plot, which can be easily overlaid with the UV chromatogram. The Rh measurements can be aver- aged across each peak and molecular weight estimate deter- mined by using the Concentration Calculator utility (right) built into the pUNk software.

Data reproduced by kind permission of Meindert Lamers, MRC Laboratory of Molecular Biology, Cambridge, UK.

Waste

Flow cell tubing IN

Flow cell tubing OUT

SEC platform

Injection Valve

Technical Note

13

Measuring the Sub-Nanometer Range with the pUNk Background: Caffeine is a bitter tasting substance with well-known physiological properties, which stimulate the mind and body in numerous ways. Having a molecular weight of 194.2 g/mol, it is a very small molecule, which readily dissolves in water. The characteristics of caffeine make it suitable to demonstrate the power of the pUNk dynamic light scattering system from Unchained Labs, to detect molecules or particles of less than 1nm in size. Sample Preparation: A solution of 1.4% w/v caffeine (Sigma Aldrich C0750) in water was prepared and filtered through a 0.02μm filter into a square quartz glass cuvette. Measurement: The prepared cuvette was placed into the temperature controlled sample holder of the pUNk, and allowed to stabilize at 20°C for 10 min. A single acquisition, averaged over 300s was made. Results: The light scattering signal from the dissolved caffeine is sufficient to produce a good quality correlogram with an intercept >0.2 and highly reproducible data with low polydispersity.

The mean radius was consistently reported at about 0.48nm and molecular weight estimate around 400Da indicating concentration dependent self-association into dimers, a known characteristic of caffeine in aqueous solutions. Summary: The pUNk DLS system is easily capable of discerning molecules or particles of less than 1nm in diameter, even at low concentrations.

Technical Note

14

DLS Rules of Thumb SIGNAL / NOISE

Ultimate sensitivity is determined by signal to noise ratio. Look for at least 150% of the clean water count rate (CWCR) before collecting data. Typical CWCR, 17k/s (quartz), 30k/s (rUNt cuvette) OPTIMIZE COUNT RATE

Use the intensity control slider to achieve 300 ~ 500k/s. Less than this is acceptable but consider increasing acquisition time for count rates below 100k/s. More than 2-3000k/s and the (expensive) photon counter begins to overload. OPTIMIZE ACQUISITION TIME

Try to collect at least 1M (signal) photons in total. For count rates of 500k/s just a few seconds is enough. Near the lower limit try to extend acquisition times. STABLE COUNTS GOOD

The best quality DLS result from very stable count rates throughout the acquisition period. +/- 20% variation is OK. +/-50% variation or more... BEWARE!

HIGH, STABLE COUNTS = GREAT! LOW, STABLE COUNTS = GOOD! HIGH, UNSTABLE COUNTS = BAD!* LOW, UNSTABLE COUNTS = GIVE UP?*

*Count rate instability is often caused by aggregation; so don't be disheartened if the count rate is highly unstable. The DLS system may not produce repeatable results but it is still telling you something valuable about your sample... CHECK the PdI

A low polydispersity index, PdI tells you the sample has a narrow size distribution: < 0.1 means highly monodisperse (e.g. certified standards)

0.1 ~ 0.25 means a narrow distribution with few aggregates >0.25 polydisperse with significant aggregate content

DISREGARD SMALL PEAKS

Intensity distribution peaks in the >1000nm size range having less than about 5% total area can generally be disregarded (baseline noise). Peaks of less than 0.3nm can also be ignored (intrinsic/solvent noise).

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