ProLinC: Sample requirements

Absorption/fluorescence Spectroscopy

Various plate-reader based instruments are available, all capable of using standard microplates from 6 to 1536-well. Plates must comply with ANSI/SLAS standards.

TECAN Infinite M1000 Pro; BMG FluoStar Galaxy; BMG Clariostar; Thermo Scientific Varioskan Flash; Varian Cary Bio 100 EL00093134 series II

  • High level of purity of the fluorophore is required, ideally with distinct polarization properties compared to any intrinsic fluorescence of the sample. Impurities or competing absorbances will affect sensitivity and accuracy of readings.
  • Generally, less than 100 uL of material is sufficient @0.2 uM to perform a standard enzymatic assay.

Analytical ultracentrifugation

Beckman Coultier Optima XL-I Analytical Ultracentrifuge

The technique relies heavily on selective absorbance measurements to monitor sedimentation effects so high levels of purity is not an absolute concern. This does become a factor of consideration if a specific absorbance wavelength is not selectable for the target protein in an impure mixture.

Circular Dichroism spectrophotometry

Applied photophysics Chirascan

We have access to 1 mm (350 ul) and 5 mm (1750 ul) cuvettes. Note that recommended sample concentration is inversely proportional to the pathlength and the molecular weight! (e.g. for a 10kDa protein: 20 uM for 1mm and 4 uM for 5 mm cuvettes respectively).

  • Samples need to be highly pure, free from aggregates and their concentration known.
  • Buffer (ideally phosphate) should be as dilute as possible and contain only minimal concentrations of salt or additives to avoid undue absorbance.

Differential Scanning Calorimetry (DSC)

Calorimetry sciences corporation N-DSC III

  • Pure samples are required… impurities will lead to confusing and potentially misleading results.
  • Sample and ‘blank’ need to be perfectly ‘buffer matched’ – recommended to perform dialysis to deliberately match sample and blank.
  • Expect to use >600 ul per run of each sample and blank buffer
  • Minimum concentration is 1 mg/ml; recommended to use 2 mg/ml.
  • It should be noted that a typical experiment will take a complete day (over night with the blank!).

Differential scanning fluorimetry (DSF)

CFX Connect™ Real-Time PCR Detection System

  • Protein samples need to be pure – denaturation (thermal stability) is monitored via access of the dye ‘cypro orange’ to the exposed hydrophobic surfaces of proteins… including impurities!
  • When assaying thermal stabilisation properties of small molecules, it is crucial that these are also pure, and their concentrations accurately known.
  • Typically use 1 mg/ml starting stock of protein… 10 ul diluted to 25 ul per well used (with dye and any test reagent, buffer, compound or additive etc. to be assessed).

Dynamic Light Scattering

Wyatt DynaPro PlateReader-II

The technique and instrument can be used in two distinct ways: 1) particle characterisation and 2) sample quality assessment. Aggregates will dominate and interfere with any scattering measurements, so it is important to centrifuge or filter samples (0.22 microns) prior to measurements. 96, 384 and 1536-well plates are available.

  • Pure samples are preferred in general but for quality assessment any sample can be used. The method will then indicate how good or bad the ‘mixture’ or sample is – impurity can be many forms… heterogeneity in oligomerisation or conformation, or contaminants. Clearly for particle characterisation, purity is paramount otherwise results will be misleading – if any can be produced.
  • Sample volume is 20 (384) down to 4 ul (1536 well plate)
  • Minimum recommended concentration is 0.1mg/ml.
  • Concentration requirement is inversely proportional to molecular size; a general recommendation is 0.2 mg/mL (20 uM) for a 10-kDa species.
  • It measures particle and molecule size, from 0.5 nm to 1 um (1-1000kDa).

Fluorescence Microscopy

Spectraview Epi-fluorescence microscope Leica DM 6000B

  • Epifluorescence microscope with hyperspectral imaging head.

Zeiss Confocal fluorescence microscope Laser Scanning Microscopy 780

  • An inverted Zeiss (Axio Observer.Z1) LSM 780 confocal laser scanning microscope system equipped with a 32 channel QUASAR GaAsP spectral array detector, a tuneable laser (488–640 nm) and a modular fluorescence lifetime imaging (FLIM system) from Becker and Hickl.
  • The microscope system can be utilized for conventional confocal imaging, as well as hyper-spectral imaging and FLIM.

Isothermal titration calorimetry

All samples and reagents must be pure, any impurities will affect binding kinetics. This includes the presence of any aggregates. This is important for both the titrant (be-it protein or small molecule) and sample – centrifuge or filter samples before use. Use degassed buffers and avoid additives and reducing agents. If a reducing agent is required for protein stability, it is recommended to use TCEP <5 mM, although it should be noted TCEP is not stable in phosphate buffers.

Concentrations should be well defined. For accurate results it is critical that the titrant concentration in particular, is accurately determined.

It is also critical that the buffer components of the sample cell and the titrant in the syringe are matched precisely. Any differences between the components will lead to high ‘heat-of-dilution’ effects which may mask titration results.

General guideline is the titrant in the syringe should be 20x expected kd of interaction.

Microcal VP-ITC

  • Typically uses 1.75 ml of 10-200 uM protein sample in the sample cell and 400 ul of 0.1-1 mM of titrant.

Malvern PEAQ-ITC

  • Sample usage 350 ul of 10-400 uM
  • Active syringe volume 40 ul of 0.1-1 mM of ligand.

Microscale Thermophoresis

Nanotemper Monolith NT.115 (Green/Red)

Amine coupling and NTA (His-tag) labelling methodology is available. MST optimized assay buffer (50 mM Tris pH7.4, 150 mM NaCl, 10 mM MgCl2, 0.05% Tween-20); and PBS with 0.05 % Tween-20; along with a variety of capillaries are available for titration experiments.

  • As a general consideration all samples should be pure; and concentrations precisely known.
  • The amine labelling is more critical of purity than the His tag method since all primary amines will be labelled including those on any contaminants.
  • Amine-based labelling should be carried out in a buffer that does not contain primary amines (in addition, it should be noted that reducing agents will affect the labelling efficiency). Typically, at least 100ml at 20 uM is needed.
  • For NTA ‘His tag’ based labelling, one should also avoid EDTA, imidazole and metals ions. A typical labelling experiment requires 100 ul at 200-800nM (depending on the NTA affinity to the His tag). The dye only labels His tags and so this labelling procedure can accommodate some level of impurity and the ‘labelled’ material does not need purifying.

One experiment uses 160 ul of labelled ‘target’ protein @20-200 nM, and 20 ul of titrant (with a concentration >40-fold above the expected KD).

Multi-angle static light scattering with size exclusion chromatography

Wyatt DynaPro Nanostar/miniDAWN TREOS/Optilab T-rEX connected to HPLC system

For meaningful results sample purity must be exceptional. Any impurities with a similar molar mass and/or radius of gyration will affect the molar mass determination. A general recommendation is that samples have previously passed through a size exclusion column and demonstrate single-band priority by SDS-PAGE.

Currently, there are 30, 75 and 200 increase columns available, with the same 10/30 geometry.  The instrument has a proven sensitivity down to 10 ul @25 uM for BSA (the trimer species is identifiable, but its characterisation is not truly accurate); as a rule, 10-100 ul sample at >50 uM is optimal. 

Nano Differential Scanning Fluorimetry

Prometheus NT.48 (Nano Temper)

We have the capability to run melting experiments to 110ºC.

For meaningful results sample purity must be exceptional. This technique uses the intrinsic fluorescence of tryptophan residues in protein and changes due to shifting environment. during unfolding, any contaminating tryptophan signal will obscure these effects.

Proteins of interest need to contain tryptophan and/or tyrosine residues – tyrosine gives a reduced signal!

  • Concentrations of protein (tryptophan!) should be from 0.015-1.5 mM.
  • Minimum volume is 5 ul per sample – it is generally recommended to run in triplicates -> 15 ul.

Small Angle X-ray Scattering

Anton Paar SAXSess

  • Well behaving material is paramount to maximise success: general recommendation that protein homogeneity and mono dispersity should be demonstrated prior to any SAXS measurement.
  • Minimal volume: 20 ul.
  • 1-10 mg/ml concentration series is recommended for complete analysis.
  • Blank and sample buffers must be ‘buffer matched’.

Surface Plasmon Resonance

GE Healthcare Biacore T200

Three traditional SPR running buffers are available:- 10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005 % Tween-20 (HBS); 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.005 % Tween-20 (TBS); and (10.1 mM Na2PO4, 1.8 mM KH2PO4 pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.005 % Tween-20 (PBS). There is also access to coupling reagents and buffers if sensor chip design and development is required.

  • Pure samples are required. This is absolutely critical for the amine-coupling method and for all analytes to be tested. (Possibly not as critical for ‘ligand’ NTA capture.
  • For ‘Ligand’ immobilization ~10-50 ug (~200 ul) of a protein is generally required.
  • ~300 ul of the analyte is needed at 10x expected Kd.
  • For best results a 100-fold range of ‘analyte’ concentrations should be attempted 0.1–10 x Kd.
  • Concentration of the analyte needs to be determined as accurately as possible.
  • Analytes need to be larger than 150 Da.
  • Running buffer and analyte buffers should be matched – particularly crucial for small molecule and when DMSO is present.