EMBL-SPC: Sample requirements

1. Biolayer Interferometry (BLI) – Octet RED96

Sample Requirements

Sample type: Proteins, antibodies, peptides, media containing serum, buffers containing DMSO, periplasmic fractions, untreated cell culture supernatants, and crude cell lysates
Measures Ka, Kd, K_D
K_D range: < nM -mM
Sample limits: analyte >200 Da
Sample volume: 180–220 μL/well per measurement of load and analyte (up to 8 at once)
Sample concentration: Load: 1-50 µg/ml; Analyte: 0.1-10 K_D
Biosensors can be requested to the SPC corefacility. Please check with us in advance.

2. Surface Plasmon Resonance (SPR) – GE/Cytiva Biacore T200

Sample Requirements

Sample type: LMW drug candidates to high molecular weight proteins (also DNA, RNA, polysaccharides, lipids, cells, and viruses) in various sample environments (e.g., in DMSO-containing buffers, plasma, and serum).
Measures Ka, Kd, K_D
K_D range: < nM -mM
Sample limits: analyte >150 Da
Sample volume per experiment: ~200 µl per immobilization and ~300 ul for each analyte concentration.
Sample concentration: Load: 1-50 µg/ml Analyte: 0.1-10 K_D
Chips and other consumables can be requested to the to the SPC corefacility. Please check with us in advance.

3. Isothermal Titration Calorimetry (ITC) – Microcal VP

Sample Requirements

Sample type:Characterization of molecular interactions established by: Protein-small molecule ( lipids, sugars, peptides, compounds), Enzyme-inhibitor, Antibody-antigen, Protein-protein, Protein-DNA, protein-Metal ion-, assessment of biological activity, Enzyme kinetics.
Active sample cell volume 1.4mL (2mL total for filling sample cell)
Active syringe volume 300 µL (500 µL total for filling the syringe)
K_D range: < nM –mM
An ITC experiment consists of two titrations: titrant (syringe) injected to the macromolecule (sample cell) and titrant (syringe) injected to the buffer (sample cell).
An example of a preliminary experiment is to inject aliquots of the titrant (300 µL at concentration 10 times higher than the protein, 100 µM-1mM) into the sample cell containing the protein solution (1.4mL, at 10-100 µM)
Molar concentration should be accurately measured. Errors in Cell concentration affect stoichiometry. Errors in the Syringe concentration will directly translate to errors in the K_D, and affect ΔH and n. Aim for a c value between 10-100 for optimal fit.
No buffer or salt limitations, except DTT as reducing agent and unstable chemicals. Avoid β mercaptoethanol, prefer to use TCEP (except: TCEP is not stable in Phosphate buffers). DMSO has high heats of dilution and should be matched extremely well between the cell and the syringe.
The two binding partners must be in identical buffers to minimize heats of dilution which can mask heats of binding.
Additional buffer (matched to samples) is required for baselines, dilutions and rinsing the cell.
Using degassed buffers will reduce the introduction of air bubbles and improve results.
Protein aggregates will interfere with ITC: Centrifuge or filter samples before use. Assess protein heterogeneity via light scattering. Purify protein samples with soluble aggregates by size-exclusion chromatography.
The technique does not require any labeling or modification of materials. ITC accuracy is not affected by ligand and protein sizes.

4. Isothermal Titration Calorimetry (ITC) – Microcal PEAQ-ITC

Sample Requirements

Sample type: Characterization of molecular interactions established by: Protein-small molecule ( lipids, sugars, peptides, compounds), Enzyme-inhibitor, Antibody-antigen, Protein-protein, Protein-DNA, protein-Metal ion-, assessment of biological activity, Enzyme kinetics.
Active sample cell volume 200 µL (350 µL total for filling sample cell)
Active syringe volume 40 µL (75 µL total for filling the syringe)
K_D range: nM-µM
An ITC experiment consists of two titrations: titrant (syringe) injected to the macromolecule (sample cell) and titrant (syringe) injected to the buffer (sample cell).
An example of a preliminary experiment is to inject aliquots of the titrant (40 µL at concentration 10 times higher than the protein, 100 µM-1mM) into the sample cell containing the protein solution (200 µL, at 10-100 µM)
Molar concentration should be accurately measured. Errors in Cell concentration affect stoichiometry. Errors in the Syringe concentration will directly translate to errors in the K_D, and affect ΔH and n. Aim for a c value between 10-100 for optimal fit
No buffer or salt limitations, except DTT as reducing agent and unstable chemicals. Avoid β mercaptoethanol, prefer to use TCEP (except: TCEP is not stable in Phosphate buffers). DMSO has high heats of dilution and should be matched extremely well between the cell and the syringe.
The two binding partners must be in identical buffers to minimize heats of dilution which can mask heats of binding.
Additional buffer (matched to samples) is required for baselines, dilutions and rinsing the cell.
Using degassed buffers will reduce the introduction of air bubbles and improve results.
Protein aggregates will interfere with ITC: Centrifuge or filter samples before use. Assess protein heterogeneity via light scattering. Purify protein samples with soluble aggregates by size-exclusion chromatography.
The technique does not require any labeling or modification of materials. ITC accuracy is not affected by ligand and protein sizes.

5. MicroScale Thermophoresis (MST Label Free)

Sample Requirements

Sample type: The applications range from small-molecule binding events to protein-protein interactions and interactions of multi-protein complexes.
Sample limits: from 100 Da to 1 MDa
K_D range: nM –mM
Sample volume per experiment: 10 μl/capillary.
Sample volume and concentration per experiment:
Target Sample (with intrinsic protein fluorescence) at ~ 0.5 µM to 5 µM and a volume of 200 µL/titration.
Ligand Sample (the non-fluorescent binding partner): ~ 50 µL/titration, at 2X working concentration (bring the highest stock concentration available for an unknown K_D). Recommended working concentration ≥ 50X K_D
Capillaries can be provided by the SPC Core Facility.

6. MicroScale Thermophoresis (MST NT.115 with Blue/Red detectors)

Sample Requirements

Sample type: The applications range from small-molecule binding events to protein-protein interactions and interactions of multi-protein complexes.
Sample limits: from 100 Da to 1 MDa
K_D range: nM –mM
Sample volume per experiment: 10 μl/capillary.
Sample volume and concentration per experiment:
Target Sample (for labeling the protein) at ~ 5-20 µM. Concentration of labeled molecule: 10 nM to 200 mM. Volume of 200 µL/titration.
Ligand Sample (the non-fluorescent binding partner): ~ 50 µL/titration, at 2X working concentration (bring the highest stock concentration available for an unknown K_D). Recommended working concentration ≥ 50X K_D
Capillaries and labeling kits can be requested to the SPC corefacility.Please check with us in advance, specially for the availability of the kits.

7. Nano Differential Scanning Fluorimetry (nDSF)

Sample Requirements

Sample type: any biological sample, from soluble proteins to integral membrane proteins, Any other type of samples such as serum or cell lysate and other additives/detergents are can be analyzed in the nanoDSF setup. Also, almost any buffer, additives and detergents can be measured.
Sample limits: From 1 kDa to 1 MDa
Sample volume per experiment: 10 μl/capillary.
Sample condition: Protein with intrinsic fluorescence reports (tryptophans and/or tyrosines)
Sample concentration: Wide concentration range:from 5 µg/ml to 200 mg/ml
Very short analysis time: enables high throughput up to 48 conditions
Optimal data quality and resolution: Dual 350/330 nm fluorescence detection
Wide temperature range: Analysis possible from 15°C to 95°C
No labeling required: Close-to-native analysis possible
Capillaries can be requested to the SPC corefacility.

8. Dynamic Light Scattering (DLS) – Cuvette-based DLS Instrument

Sample Requirements

Sample types: DLS is used to characterize the size/monodispersity of various particles including proteins and micelles. DLS is suitable for ensemble measurements ranging from hydrodinamic ratios ( Rh) values of 0.2 nm up to 5,000 nm.
Sample volume per experiment: 10 μl on cuvette.
Buffer (matched to samples) is required.
Aggregates will interfere with DLS measurements: Centrifuge or filter samples and buffers (0.22 microns or 0.11 microns) prior to the measurements.

9. Circular Dichroism spectroscopy

Sample Requirements

Sample types: Circular Dichroism (CD) Spectroscopy is used to determine the optical isomerism and secondary structure of molecules. Proteins have many chiral centers. CD spectra in the Far-UV region (185 – 250 nm) can be used to determine protein secondary structure. Characteristic peaks for Thermal stability (Tm) can be measured by following changes in molar ellipticity with increasing temperature.
Sample concentration and volume: An accurate protein concentration is required for all CD experiments. The amount of sample required for a CD experiment depends on the size, cuvette path length and type of measurement being performed.
1 mm pathlength cuvettes hold 300 µl and a 1 cm cuvette holds 3 ml.
Required protein concentration is inversely proportional to the cuvette pathlength, and thus a 1 mm path cuvette requires 10x the concentration of a 1 cm cuvette.
Recommended concentrations for Far-UV measurements of protein secondary structure are:
0.2 mg/mL in 1 mm path cuvette
0.02 mg/mL in 10 mm path cuvette
Mol/L=115/(MW*7000)*10/pathlength(mm).
For alpha-helical proteins you may need ½ this concentration. For beta-sheet proteins, you may need double the concentration. If possible, make a concentrated stock solution of your protein (at least 2X) and dilute as needed.
Protein aggregates can interfere with the CD signal: Assess protein heterogeneity via light scattering and purify protein samples with soluble aggregates by size-exclusion chromatography.
Samples can be recovered from the cell. This is not recommended for thermal melts, unless you know your thermal denaturation is reversible.
Buffer selection: Buffer selection is critical for accurate CD measurements. Solvent absorbance can severely interfere with the CD signal. Many commonly used buffers and additives absorb in the far UV region used for CD measurements. The ideal CD experiment is performed in a buffer in which your protein is well behaved and soluble with no buffer absorbance through the range of the CD spectrum.

10. Mass spectrometry

We offer the scientific community a service of analysis and characterization of proteins and provides the following techniques:

High-resolution, exact mass determination of proteins and peptides
Study of protein complexes by native mass spectrometry.

Our instrument is a MALDI TOF / TOF MS (Autoflex maX, Bruker Daltonics) with the following characteristics:

ion source: MALDI
analyzer: TOF (linear mode and reflectron)
m / z up to 500,000 (linear mode)
resolution: up to> 26,000 ( reflectron mode) and> 2’500 (linear mode)
Connected to a CovalX HM4 High-Mass System which enables: Protein complex analysis and Intact Protein Analysis (up to 2MDa)

Sample Requirements

Use high quality water for all solutions (HPLC or MilliQ quality)
Buffers and additives: avoid phosphate (PBS) and sulfate buffers, detergents (eg Triton, CHAPS…) and contamination by polymers (e. g polyethylene glycols)
High salt/buffer concentrations can prevent matrix crystallisation. Then it might be necessary to dialyse the protein solution before
Sample concentration and volume: The drop deposited on the plate should contain around 1 pmol of protein (0.1 – 10 pmol/ul) (If concentration is too high, the matrix will not crystallis. Otherwise, if concentration is too low, the count rate will be too low). For general purposes, a stock of 1 uM and a volume of 5-10 uL is enough.
Calibration standards: We have these calibration standards in the facility:
* Peptides: 1 – 3 kDa
* Protein I: 4- 20 kDa
* Protein II: 10 – 70 kDa

11. Fourier Transform Infrared Spectroscopy (FTIR)

The VERTEX 70v FTIR Spectrometer provides the possibility to acquire a complete far and mid IR spectrum from 6000 cm^-1 to 50 cm^-1 in a single step measurement with no need to change any optical component. FTIR-Spectroscopic analysis of proteins can include:

Determination of structural changes
Measurement of temperature ramps
Concentration determination
Secondary structure analysis
Protein-ligand-interaction

Sample Requirements

Aquaspec cell – Sample consumption: ca. 30 ul
BioATR II cell – Sample consumption: 10 – 20 ul
Buffer selection: the buffer must be taken as reference at the beginning.

12. Refeyn One – Mass photometer

The Refeyn Onesystem applies the principle of interference reflection microscopy and interferometric scattering microscopy to quantify light scattered by a single molecule on a glass surface. The amount of light scattered by each molecule is directly correlated to its molecular mass.

Based on the above principle, the Refeyn One can monitor protein-protein interactions at a single-molecule level with high-sensitivity and can at the same time determine molecular weight of proteins and protein complexes with a high dynamic range and great accuracy.

The mass photometer is an ideal tool for quality control in the protein structural analysis workflow as it can assess the molecular mass and the oligomerisation status of a sample in one measurement.

Sample Requirements

Sample concentration and volume: The amount of protein needed for an analysis is very small, between 5µl to 20 µl of protein solution needed at a nano-molar concentration range ( e.g 100 nm). After the initial set up, measurement takes in general one minute or slightly longer if required.
Mass range: 40 kDa -5 MDa
Concentration range: 100 pM-100 nM (particle concentration)
Buffer requirements: Centrifuge buffers (0.22 microns or 0.11 microns) prior to the measurements. Avoid buffers with high percentage of glycerol.
Membrane proteins could be measured upon consideration. For further info, please read as published: https://www.sciencedirect.com/science/article/pii/S2451929420305945

13. SURFE²R N1 (SSM-based electrophysiology)

The SURFE²R N1 is designed for the measurements of electrogenic transporters (symporters, exchangers and uniporters) and pumps. Usually these proteins have low turnover rates compared to ion channels. SURFE²R technology compensates for that with a large sensor size which allows for the measurement of up to 10⁹ transporters at the same time to yield the best signal to noise ratio.

Sample Requirements

Source material: Sample preparation commonly begins either with recombinant overexpression in eukaryotic cell lines (CHO, HEK and COS-1), bacteria or yeast or with the isolation of membranes from native tissue such as heart, kidney, liver, brain, skeletal muscle or gastric mucosa from different organisms including pig, rabbit or mice. Cell free expressed transporters in nanodiscs as well as whole cells have been used for adsorption to the SSM, but usually purified samples are required.
Membrane purification: When using isolated membrane samples, after cell disruption the membranes should be purified using a sucrose density gradient centrifugation. This is especially appropriate when membranes from distinct organelles like mitochondria, ER, synaptic vesicles or lysosomes are required. It is also commonly employed for the enrichment of plasma membrane fragments. Sucrose gradient centrifugation yields a significant signal enhancement compared to non-purified samples.
Protein reconstitution: High turnover transporters usually work with membrane preparations. But they show lower signal to noise compared to reconstituted samples. The ideal sample therefore is prepared by protein purification followed by reconstitution into liposomes at high protein densities. Transporters with turnover rates below 100 s-1 should be characterized using proteoliposomes with low lipid-to-protein ratio (LPR) of 5 to 10. The LPR of successfully tested proteoliposomes range from 5 (LacY, turnover of 30 s-1) to 500 (NhaA, turnover of 1000 s-1). When using reconstituted samples it is critical to ensure that no residual detergent remains in the membrane preparation after reconstitution. Different procedures are described for detergent removal, e.g. rapid dilution or incubation with Bio-Beads. If detergent remains in the protein sample, the sensor can be destroyed during the adsorption process.
Sample Concentration: The lipid concentration has to be optimized before starting the experiments. Start with a dilution sequence of the sample and check for the signal amplitudes. For your experiments then use a sample concentration achieving high signal amplitudes with the lowest sample consumption possible. Too high concentrations as well as too low concentrations can reduce the signal amplitude. The sample can be diluted in the respective non-activating buffer directly before the measurement. Usually lipid concentrations between 0.2 mg/ml and 5 mg/ml are used. In the case of membrane preparations with unknown lipid concentration, the total protein concentration can be used as a benchmark. Typical samples are prepared with 2 mg/ml to 10 mg/ml total protein concentration. For adsorption to the SSM total protein concentrations between 0.1 and 1 mg/ml are used. Since the concentration of the protein of interest cannot be adjusted directly, the output mainly depends on the expression efficiency which, therefore, also has to be optimized.
Sample Volume: For each sensor 5 to 10 µl of the diluted sample is required. If the sample amount is critical this volume could be reduced even further. Therefore 50 to 100 µl could be enough for a rough characterization of the transporter. Depending on the measurement sequence it takes up to a few days to measure 100 µl of the sample.

14. TECAN Spark 20M multimode plate reader

Spark provides unparalleled wavelength accuracy, with a dedicated High Speed Monochromator for absorbance measurements. Together with a cuvette port and the patented NanoQuant Plate, it provides an all-in-one solution for ELISAs, low volume DNA/protein quantification and fast spectral scanning.

At the heart of the instrument are its unique Fusion Optics for fluorescence, allowing any combination of filters or monochromators on both the excitation and emission sides for every measurement. This option features variable bandwidth selection and full wavelength flexibility to provide exceptional measurement performance and speed. Spark’s multi-color luminescence module offers unparalleled flexibility for virtually any luminescence measurement, including flash, glow, BRET and laserbased Alpha Technology. The instrument’s bright field cell imaging capabilities, together with its incubator-like environmental control, enable long-term cell-based experiments and live monitoring of cell growth. The conditional workflow automation minimizes hands-on times and increases reproducibility, enabling long-term experiments with precious cell lines.

Features:

Active cooling – Yes (18° C to 42° C)
Automated cell imaging – High power LED, bright-field, 4x objective, laser-based autofocus, optical resolution > 3 µm
Environmental control – Integrated gas control module for O₂/CO₂
Reagent injectors – 1 to 2 reagent injectors, with heating and stirring capability
Barcode scanner – Integrated, left or right side
Stacker – Integrated microplate stacker with dark covers for light protection
Shaking – Linear, orbital, double-orbital
Detection Absorbance, Fluorescence intensity, FP, TRF, FRET, TR-FRET, luminescence (flash, glow, multi-color), Alpha Technology, Automated live cell imaging

Applications

ELISA
Low-volume DNA/RNA quantification
Nucleic acid labeling efficiency
Protein quantification
Reporter gene assays
HTRF, Transcreener, DLR, BRET including NanoBRET
Cell counting and viabiltiy
Confluence assessment
Cell migration and wound healing
Range :200 to 1000 nm (ABS), 230 to 900 nm (FI), 370 to 700 nm (LUM)
Time :96-well < 13 s, 384-well < 22 sec, 1536-well < 34 sec
Plate Formats: 1 to 1536 wells, NanoQuant plate, Cell Chip, Cuvettes, Roboflask
Temperature: Range Ambient + 3° C to 42°C
Light Source: Xenon flash lamp, AlphaScreen laser
Validated Applications: Live Cell Imaging, Cell Counting, Cell Confluence

15. Evolution 350 UV-Vis Spectrophotometer

Specifications

Rely on the Evolution 350 Spectrophotometer’s double beam optical design providing outstanding reliability, precision performance, and long-term stability for the most demanding applications.
Optimized performance for advanced testing requirements and peak resolution capabilities with selectable bandwidths of 0.5, 1.0, 1.5, 2.0 and 4.0 nm.
Ensure accuracy of your data with optional Calibration Validation Carousel (CVC) for automated performance verification.
Get instant measurements and excellent performance over entire wavelength range of 190-1100 nm with xenon flash lamp.
Easily go from sample to final report – with intuitive INSIGHT Software guiding you every step of the way.
Obtain results you need for quantitative analysis, scanning and kinetics applications with comprehensive software tools for data collection, processing, and reporting.
Customize even complex methods with ease using workflow-oriented application modules.

Quantitative analysis

Reliable results are an essential component of quality control analyses. From simple, single-standard comparisons to standard curves based on peak area, we have the tools to get the answers you need every time.

Choose to perform your analysis in fixed or scan mode.
Select a curve fit and standard averaging as desired.
Set minimum correlation coefficients or use concentration limits to define the requirements for your standards and samples After measurements are complete, a run chart neatly displays the data and error bars, indicating whether or not each sample measurement falls within the defined concentration range.

16. SX20 stopped – flow spectrometer

Typically used to gain an understanding of reaction mechanisms including drug-binding processes, or to determine protein structure, stopped-flow spectroscopy enables the study of fast reactions in solution over timescales in the range of 1 millisecond to hundreds of seconds. A wide range of reactions can be investigated involving, for example, protein-protein interactions, ligand binding, electron transfer, fluorescence resonance energy transfer (FRET), protein folding, as well as enzyme, chemical or coordination reactions.

In most experimental set-ups, two reagents are rapidly mixed together and then ‘stopped’ in an observation cell. The sample cell is irradiated with monochromatic light and as the reaction proceeds the change in the recorded signal, usually a fluorescence signal or absorbance at a specific wavelength, is recorded as a function of time.

Analysis of the resulting kinetic transient can determine reaction rates, complexity of the reaction mechanism, information on short-lived reaction intermediates etc. A series of stopped-flow experiments can be used to show the effect of parameters such as temperature, pH and reagent concentration on the kinetics of a reaction.

Sample Requirements/ Details

Minimum volume: For single mixing experiments a minimum of 120 µL is the recommended total drive volume. Users may choose to reduce drive volume to improve sample economy under certain experimental conditions.
Cell Pathlenghts: Standard cell (20 µL): 2 mm and 10 mm pathlengths. Rapid kinetics cell (5 µL): 1 mm and 5 mm pathlengths.
Symmetric and asymmetric mixing: Symmetric mixing uses two identically-sized drive syringes, such as the standard 2.5 mL syringes provided with the instrument. Asymmetric mixing uses two syringes of different sizes to mix a volume ratio other than 1:1. For example, a 10:1 ratio (common for refolding experiments) can be achieved using a 250 µL syringe in place of one of the 2.5 mL syringes.

17. High-throughput stability screening for detergent-solubilized membrane proteins (Analytic Selector Kit) using nDSF technology

Sample Requirements

Starting concentration: Ideally, 3 to 5 mg/ml, lower concentrations may work as well. Minimum volume of 250 ul of your sample.
2x original buffer, detergent-free with its composition (including detergent CMC). For example, if the protein is provided in 20mM NaP pH 7.5, 15mM NaCl, 5% glycerol, 0.03% DDM, the corresponding 2x buffer will be: 40mM NaP pH 7.5, 30mM NaCl. Minimum volume of 10 ml of the 2x original buffer.
2x original detergent; for example, if the protein was provided in 20mM NaP pH 7.5, 15mM NaCl, 5% glycerol, 0.03% DDM, then 0.06% solution of DDM in water will be needed. Minimum volume of 10 ml of the 2x original buffer.
Number of tryptophans and/or tyrosins in protein sequence (fluorescent reporters).
Molecular weight of protein/sample.
All samples and buffers should be properly filtered.

18. Small Angle X-ray Scattering (SAXS)

Sample Requirements

Full details of sample guidelines for this technique can be found here: https://www.embl-hamburg.de/biosaxs/sample.html