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The ActiPix™ Nano-Sizing System

INTRODUCTION

The ActiPix™ Nano-Sizing system is a benchtop setup which measures the hydrodynamic radius and diffusion coefficient for species in solution. It is ideal for use in protein production, purification, analysis and crystallization labs. Key features and benefits include:

Low sample requirement—the system requires less than 1 µL for analysis thus saving precious material. A low volume nanolitre injector valve is used for sample injection.
Large dynamic range—can determine hydrodynamic radii from low to high concentrations—no pre-treatment required.
Ideal for membrane proteins—the ActiPix™ Nano-Sizing System uses UV absorbance of the species to determine hydrodynamic radius. As detergents are invisible to UV light, difficult samples such as membrane proteins are easy to characterise.

DESCRIPTION OF OPERATION

The ActiPix™ Nano-Sizing System shown in uses a novel approach to measure the hydrodynamic radius for proteins. It utilises Paraytec’s award winning UV area imaging technology. The technique allows proteins to be used in their native form, without any labelling or denaturation. A plug of protein solution is injected into a fused silica capillary via an injector valve, driven through the capillary by application of pressure via a syringe pump, and detected using ActiPix™ UV area imaging technology as it passes imaging windows at the entrance to and exit from a loop in the capillary.

THEORY BEHIND APPROACH

The ActiPix™ Nano-Sizing System uses a looped capillary. A plug of sample is injected into a low volume nanolitre valve and driven along the capillary by application of external pressure from the syringe pump. Ultraviolet (UV) absorbance of the protein zone is recorded during the first and second passes through the imaging area, giving two peaks. Whilst the peak areas are the same, since area is proportional to the amount of protein injected, the widths are different: the second peak has a greater width and lower amplitude due to Taylor dispersion. Taylor dispersion analysis (TDA) provides an absolute, simple and rapid method for determining diffusion coefficients and hydrodynamic radii.

In the ActiPix™ Nano-Sizing System, UV area imaging and two windows allow data to be collected extremely quickly in a single experiment. The peaks are fitted with an appropriate fitting function using Paraytec’s ActiPix™ Nano-Sizing Software. The area under the peak corresponds to the amount of protein injected. The times and variance of the two peaks are used to calculate the hydrodynamic radius of the protein.

The ActiPix™ Nano-Sizing software presents the data in a convenient format. An example of the data output is given in the images above.

Table 1 shows typical data obtained for bovine serum albumin (BSA) using Taylor dispersion analysis. It can be seen that the hydrodynamic radius obtained is independent of sample concentration.

Table 1: Data for BSA over the concentration range 0.05-2 mg mL-1

Concentration (mg mL-1) Hydrodynamic radius (nm)

0.05 3.26 ± 0.16

0.25 3.22 ± 0.08

0.50 3.64 ± 0.11

1.00 3.35 ± 0.05

2.00 3.39 ± 0.06

For a more detailed explanation of the theory behind the ActiPix™ Nano-Sizing system, go to our downloads section for the following publications:

ORDERING INFORMATION

Part number: ACTINSIZE

Description

Benchtop Nano-Sizing system with low sample requirement contains:

ACTID100 system
Low volume syringe pump
ActiPix™ sizing cartridge
254 nm filter
Low volume injector valve
10 m of fused silica capillary, 200 µm OD, 75 µm ID
Coupling stand
250 µL syringe for buffer delivery
Syringe for low volume injection
Fittings kit
USB data cable
Tool kit
Powerful applications software to enable sizing determination of single species, e.g. proteins in solutions

(System excludes power cables for local use)

Paraytec Ltd