The Protein Analysis Unit provides the following services:
- Biomolecular interaction analysis: (Biacore), Isothermal Titration Calorimetry (ITC) and Microscale Thermophoresis (MST).
- Protein characterization: Differential Scanning Calorimetry (DSC) as well as Differential Scanning Fluorimetry (DSF).
- Detection, Quantification and Isolation of large particles: NanoSight and Field-flow fractionation (FFF).
The services are provided to scientists at the Weizmann Institute as well as companies and other academic institutes.
Biomolecular interaction analysis
This biosensor technology is based on the principle of surface plasmon resonance (SPR). SPR is a non-invasive optical measurement technique which detects the mass-concentration of biomolecules in close proximity to the specifically prepared surface. SPR is a powerful technique for measuring binding between all types of molecular interactions. This includes drugs and targets, antibodies and antigens or any pair of interacting molecules including protein-protein, protein-nucleic acid and protein-lipid interactions. Interactions are measured in real time allowing relative comparisons between different molecules or determination of kinetic parameters. SPR does not require any type of labelling of the test molecules. All Real time kinetics data are accomplished on the BIAcore T200 instrument. Quantitative information, such as kinetic parameters and equilibrium constants for complex formation can be obtained within a range of 10-4 to 10-12M . The Biacore system characterizes molecules in terms of their:
- Specificity of their interactions
- On and off rate constants (kinetics)
- Binding strength (affinity)
Isothermal Titration Calorimetry (ITC)
ITC is an analytical tool that measures the binding affinity and thermodynamics between any two biomolecules. Binding constants in the range of 10-3 to 10-9 M can be measured.
ITC is considered the “gold standard” assay for binding, and has many advantages:
- Directly measures heat change associated with binding
- Label-free - uses native materials
- True in-solution technique
- Requires minimal assay development
- Has no molecular weight limitations
- Versatile, can be used with any biomolecule - proteins, nucleic acids, small molecule drugs, lipids, etc.
- Can be used with a wide range of biological buffers, ionic strengths, pH’s
In a single ITC experiment, one can determine:
- Binding affinity - KD (Directly measure sub-millimolar to nanomolar)
- Extend affinity range to picomolar using competitive ITC method
- Number of binding sites
- Multiple and different binding sites
- Enthalpy (ΔH) and entropy (ΔS) of binding
Microscale Thermophoresis (MST)
Microscale thermophoresis (MST) is a technology for biomolecules interaction analysis. MST is a rapid and precise method to quantify biomolecular interactions in solution at microliter scale. Microscale thermophoresis is the directed movement of particles in a microscopic temperature gradient. Any change of the hydration shell of biomolecules due to changes in their structure/conformation, results in a relative change of movement along the temperature gradient and is used to determine binding affinities. MST allows measurement of biomolecule interactions (small molecules, DNA, RNA, proteins, peptides, sugars, lipids, ribosomes etc.) under close-to-native conditions directly in solution. The measurement can be performed in a solution of choice, ranging from standard buffers to complex bioliquids including blood serum or cell lysates. One of the reactants should be fluorescently labeled. MST instrument Monolith NT.115 enables reliable measurments suitable for KD values from 10-3 to 10-12 M. .
Biophysical characterization of biomolecules
Differential Scanning Calorimetry (DSC)
DSC continuously measures heat capacity of a system as a function of temperature allowing for simultaneous determination of enthalpy, entropy and Gibbs energy for a thermal transition. This information has proven to be extremely important in the characterization of protein folding and oligonucleotide conformation.
Differential Scanning Calorimetry (DSC) is a powerful analytical tool which directly measures the change in heat associated with the unfolding of a protein, lipid, or nucleic acid. In DSC, as the biomolecule is heated at a constant rate, a detectable heat change associated with thermal denaturation can be accurately measured. DSC experiments are:
- Label-free and use native materials
- True in-solution technique
- Easy to perform - require minimal assay development
- Conducted using a wide range of biological buffers, ionic strengths and pH’s
- Universal assay – measures the heat change associated with denaturation
- Non-optical – unaffected by colored or turbid samples
- Versatile - can be used with proteins, nucleic acids, lipids and other biomolecules
In a single DSC experiment, one can determine:
- Transition midpoint (Tm)
- Enthalpy (ΔH) and heat capacity change (∆Cp) associated with unfolding
Differential Scanning Fluorimeter (DSF)
NanoDSF Prometheus NT.48 is an advanced Differential Scanning Fluorimeter for measuring ultra-high resolution protein stability using intrinsic tryptophan or tyrosine fluorescence. Applications include antibody engineering, membrane protein research, formulation and quality control.
The method is easy, rapid, and analyses stability of proteins.
The Prometheus NT.48 continuously heats up to 48 samples with an adjustable heating rate of 0.1 - 5 °C/min in a range of 20 - 95°C and simultaneously reads both the fluorescence and back reflection signal. Thus, the aggregation onset temperature can be determined for up to 48 different conditions in parallel to the melting temperature.
As no secondary reporter fluorophores are required, protein solutions can be analyzed independently of buffer compositions, with a maximal protein concentration range from more than 250 mg/ml down to 10 µg/ml. This allows for the analysis of detergent-solubilized membrane proteins as well as highly concentrated antibody formulations.
Nanosight instrument NS300 is used for particle visualization and rapid, automated analysis of size distribution and concentration of all types of nanoparticles from ~10nm to 1000nm in diamter. The instrument is used for analysis of nano particles, viruses, exosomes and liposomes.
Field Flow fractionation (FFF)
AF2000 Field-flow fractionation (FFF) instrument can separate and isolate particles over a wide size range from 1 nm up to 100 µm. This technique separates the particles present in the fluid, depending on their differing Field-Flow Fractionation is providing fast, gentle and high resolution separations of any particulate matter in a liquid medium. The separated fractions can be collected at any temperature.