Techniques

What we can measure:

In the table below is an overview of the most used functions for the different techniques. 

 

Fida 1Fida NeoClariostar PlusMonolithPrometheus PantaAkta
BindingXXXX X
SizeXX  XX
Polydispersity XX  XX
OligomerisationXX  XX
Molecular Weight    XX
Stability  X X 
AggregationXXX X 
Kinetics XX   

Fida 1 & Fida Neo

Image adapted from FidaBio.com; it shows a capillary with in yellow the pattern the sample will make based on taylors dispersion within the laminar flow (arrows) of the capillary.
Image adapted from FidaBio.com; it shows a capillary with in Yellow the pattern the sample will make based on taylors dispersion within the laminar flow of the capillary.

Fida stands for Flow Induced Dispersion Analysis.

The Fida has a long capillary shown in the picture with the detector at the end. In this capillary there is a laminar flow, which when a sample is injected causes the sample to diffuse in a specific patern. This fluorescence patern is detected by the detector and based on the fluorescent signal over time, the size (hydrodynamic radius) of the sample gets calculated.

This technique can thus be used to measure the size (hydrodynamic radius) in nm of the sample, but also to asses binding, oligomerisation and aggregation as well as polydispersity of the sample. The Fida Neo has a newer software version which also allows it to measure binding kinetics. 

For more information, please contact Ren茅e Koopman or look at the website of FidaBio.

Clariostar Plus

A schematic display of a well in a well plate which shows, from left to right, absorbance, fluorescence and luminescence
A schematic display of a well in a well plate which shows, from left to right, absorbance, fluorescence and luminescence

The Clariostar Plus is the real workhorse of our facility. 

It can measure fluorescence, Absorbance and luminescence. It has filters installed to measure Fluorescent anisotropy. It can measure kinetics and has an injector. 

This technique can thus be used to measure binding/oligomerisation and binding kinetics, but it is also used to asses aggregation with THT assays. It can also be used for ELISA screenings, Bradford assays and other OD measurements. 

For more information, please contact Ren茅e Koopman or look at the website of . 

Monolith

MST explained in a graph
Microscale thermophoresis works by heating up the sample with an IR laser. This heating up gives a difference in signal for bound and unbound proteins/fluorescent particles.

The Monolith measures binding by Microscale Thermophoresis.

Microscale thermophoresis works by heating up the sample locally with an IR laser. This heating up gives a difference in signal for bound and unbound proteins/fluorescent particles. For example the fluophore might be more stable (diffent photobleaching rate) or the speed with which the particle moves away from the heat source is different. This difference in fluorescent signal at a specific timepoint is put in a graph against the ligand concentration leading to the Kd of the binding. 

This technique can only be used to measure binding/oligomerisation, but it does give you some information on the stability and aggregation of your sample. 

For more information, please contact Ren茅e Koopman or look at the website of . 

Prometheus Panta

The principle of protein unfolding by heat explained in a figure
The prometheus panta measures unfolding of proteins by heat based on thrypthophan fluorescence.

The Prometheus panta can measure a lot of different characteristics of your sample.

Nano differential scanning fluorimetry (NanoDSF) works by measuring the changes in tryptophan fluorescence while unfolding the protein either with heat or with chemicals. This gives you insight into the stability, buffer preferences and possible binding partners of your sample. Dynamic light scattering (DLS) works by measuring the scattering pattern of a laserbeam after hitting a sample. This provides information on the size and polydispersity of your sample as well as oligomerisation and aggregation. Static light scattering (SLS) is similar to DLS but looks at the scattering pattern from a different angle, this technique can tell you the Molecular weight as well as self association constant of your sample. Lastly the Prometheus panta also measures backscattering of the sample which indicates changes in turbidity. 

For more information, please contact Ren茅e Koopman or look at the website of . 

Akta (Size Exclusion Chromatography)

Schematic display of Size exclusion chromatography where larger particles move faster than smaller particles.
Schematic display of Size exclusion chromatography where larger particles move faster than smaller particles.

Akta purification systems are not only for purifying proteins. 

At the Protein Research Centre we have a whole setup for protein purification including high speed and ultracentrifuges, Akta pure systems and Akta pure micro systems as well as SDS-page (imaging) equipment and a nanodrop to determine protein concentration accurately. 

Apart from purifying your protein the Akta systems in combination with the correct column, can give you information on the size and molecular weight of the sample, polydispersity, oligomerisation and binding events. 

For more information, please contact Ren茅e Koopman or look at the website of .