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SPR at the CMI

Surface Plasmon Resonance (SPR) is an optical technique used to measure molecular interactions in real time. SPR can occur when plane-polarized light hits a metal film under total internal reflection conditions.  SPR signal is directly dependent on the refractive index of the medium on the sensor chip.  The binding of biomolecules results in changes in the refractive index on the sensor surface.  In an SPR experiment, one molecule (the Ligand) is immobilized on a sensor chip and binding to a second molecule (the Analyte) is measured under flow.  Response is measured in resonance units (RU) and is proportional to the mass on the surface, and for any given interactant, the response is proportional to the number of molecules bound to the surface.  Response is recorded and displayed as a sensogram in real time.  SPR experiments can be used to measure kinetic binding constants (ka, kd) and equilibrium binding constants (affinity, Ka = 1/Kd).                     

       SPR Samples                                 Biacore T200                                       

The CMI has a Biacore T200 from Cytiva, formerly GE Life Sciences.

BLI at the CMI

Bio-Layer Interferometry (BLI) is an optical technique for measuring macromolecular interactions by analyzing interference patterns of white light reflected from the surface of a biosensor tip.  BLI experiments are used to determine the kinetics and affinity of molecular interactions. In a BLI experiment, one molecule is immobilized to a Dip and Read Biosensor and binding to a second molecule is measured. A change in the number of molecules bound to the end of the biosensor tip causes a shift in the interference pattern that is measured in real-time. 

BLI Samples         ForteBio Octet Red384ForteBio BLItz

The CMI has two ForteBio instruments for measuring BLI: the Octet RED384 and the BLItz.  The Octet RED384 is more sensitive and higher throughput and can be used for macromolecular and small molecule binding experiments.  The BLItz uses a very small sample volume, but is more time consuming and cannot measure molecules smaller than 10 KDa.

                 

ITC at the CMI

Isothermal Titration Calorimetry (ITC) is a label-free method for measuring binding of any two molecules that release or absorb heat upon binding. ITC can be used to measure the thermodynamic parameters of biomolecular interactions, including affinity (KA), enthalpy (ΔH), entropy (ΔS), and stoichiometry (n). Energetically favorable binding reactions have negative free energy values, ΔG = RTlnKD. ΔG, has two energetic components, enthalpy (ΔH) and entropy (ΔS) and their contributions are expressed as: ΔG = ΔH -TΔS. In an ITC experiment, ΔH of binding is measured directly. The microcalorimeter has two cells: one contains water and acts as a reference cell, the other contains the sample, into which a binding partner is titrated using an injection syringe. Heat sensing devices detect temperature differences between the cells when binding occurs in the sample cell and give feedback to the heaters, which compensate for this difference and return the cells to equal temperature.

Heats measured in an ITC are a combination of heats of binding and heats of dilution, so a control experiment to measure heats of binding must be performed. Therefore, one experiment includes two titrations:

  • Component A (in the syringe) injected into component B (in the cell).
  • Component A (in the syringe) injected into buffer (in the cell).
ITC schematic

The CMI has a Microcal ITC200 from Malvern

MST at the CMI

MicroScale Thermophoresis (MST) is an immobilization-free technology for measuring biomolecular interactions. The MST instrument detects the motion of fluorescent molecules along a microscopic temperature gradient, which reflects changes in the molecular hydration shell, charge, or size. Since one or all of these parameters will change with virtually every binding event, a wide range of biomolecules can be measured, from ions and small molecule fragments to large macromolecular complexes, in small volumes (~20 μl), in a wide range of standard buffers and complex mixtures such as liposomes, detergent, serum, and cell lysates.

MST Theory

 

The CMI has a Monolith NT.115pico from NanoTemper Technologies.

SEC-MALS at the CMI

The CMI has a SEC-MALS system with a Wyatt Dawn Heleos Multi-Angle Light Scattering (MALS, 18-angle) Detector, with in-line DLS detector and Optilab TrEX refractive index detector.  SEC-MALS is used to measure the weight-averaged molar mass of proteins (and other macromolecules) in solution, and can determine oligomeric state and sample polydispersity.  By using two concentration detectors (RI and UV), the molar mass and weight-fraction of a modifier can be determined. This can be used to measure protein and modifier masses of integral membrane proteins in detergent micelles and of glycosylated proteins.

Wyatt sec-mals system

DLS at the CMI

The CMI has Wyatt DynaPro Plate Reader III.  The DynaPro Plate Reader III measures dynamic light scattering (DLS) and Static Light Scattering (SLS) at high-throughput on many sample types in standard microwell plates. and can be used to rapidly assess protein quality in solution.  DLS measurements of hydrodynamic radius allow the monodispersity and aggregation state of a sample in solution to be measured in minutes and can be used to monitor protein quality during any (or all) stages of a protein purification project, or after storage.  DLS can also be used to size a variety of nanoparticles, including liposomes, micelles, bicelles, and nanodiscs and for aggegation analysis of chemical compounds. 

dynapro plate reader III

CD at the CMI

Circular Dichroism (CD) Spectroscopy is used to determine the optical isomerism and secondary structure of molecules. Circular dichroism (measured in molar ellipticity) is the difference in absorption of left-handed and right-handed circularly polarized light and can be observed in optically active molecules with chiral centers. 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.

CD Sample Data

The CMI has a Jasco J-815 CD Spectropolarimeter with a Peltier temperature controller and single cuvette holder.

DSF at the CMI

Differential Scanning Fluorimetry (DSF) measures protein unfolding by monitory changes in fluorescence as a function of temperature. Conventional DSF uses a hydrophobic fluorescent dye that binds to proteins as they unfold. NanoDSF measures changes in intrinsic protein fluorescence as proteins unfold.

DSF Theory

The CMI has a modified Life Technologies Quant Studio 6/7, for conventional DSF.

The CMI has a Prometheus NT.Plex instrument from NanoTemper Technologies with aggregation optics. The CMI has these Data collection and analysis software packages: PR.ThermControl for thermal stability data collection, PR.ChemControl for chemical stability data collection, PR.TimeControl for time interval data collection, and PR.Stability Analysis for advanced data analysis.

 

SPR Resources

Supplies Provided by User:

  • Cytiva Biacore Series S sensor chips
  • running buffer (should include some detergent, such as 0.05% Tween 20)
  • all reagents and samples for immobilization and binding assays
    • all concentrations of analyte sample should be in running buffer (with fixed concentation of all components, including DMSO, detergent, buffer and salt concentrations)
  • pipettors for sample and reagent liquid handling
  • (optional) 96-well and 384-well reagent plates and foil

Supplies provided by the CMI:

  • plastic tubes and caps (3 sizes)
  • instrument cleaning reagents: Desorb 1, Desorb 2, Biadisinfectant
  • Purified water for instrument cleaning and running in Standby mode

CMI Biacore T200 SPR Getting Started Guide

Other Resources:
Biacore T200 Getting Started Handbook, an intro to SPR and tutorial, from Cytiva, formerly GE Healthcare.
Biacore Sensor Surface Handbook, a guide to SPR immobilization strategies, from Cytiva.
Biacore T200 Product Information from Cytiva.
Purchase Sensor chips and reagents from Cytiva.  

SPR Pages, a good resource for all things SPR-related.

Additional Biacore T200 instrument and software manuals are available to users on the Biacore T200 computer.

BLI Resources

Octet RED 384 Supplies

User-Provided Required supplies:

  • ForteBio Dip and Read sensors
  • Black 96-well plate to soak sensors
  • Black 96-well or 384-well plate for samples and reagents
  • Pipettors for sample handling

Optional provided supplies and equipement:

  • Empty BLI Sensor trays to use as working tray during experiment
  • 8-sensor transfer tool
  • Refrigerated centrifuge with microplate rotor

BLItz Supplies

User-Provide Required supplies:

  • ForteBio Dip and Read sensors
  • 96-well plate to soak sensors

CMI-Provide Required Supplies:

  • Black 0.5 ml tubes (provided by CMI)

 

Popular ForteBio Dip and Read Biosensors for Kinetics* Part Number
Streptavidin (SA) biosensors 18-5019 (96/tray)
High Precision Streptavidin (SAX) biosensors 18-5117
Super-Streptavidin (SSA) biosensors (for small molecules) 18-5057
anti-His (HIS1K) biosensors 18-5120
Ni-NTA (NTA) biosensors 18-5101
Anti-Human IgG Fc biosensors 18-5010

* See ForteBio website for more sensor types

OCTET Black Microplates** Part Number
Greiner Bio-One 96-well black flat-bottom PP, 200 µL 655209 (VWR 82050-784)
Greiner Bio-One 384-well black flat-bottom PP, 80-120 µL 781209 (VWR 82051-318)
ForteBio 384-well black tilted-bottom PP, 60 µL 18-5080

** Only Greiner Bio-One brand, black microplates or ForteBio plates are recommended by ForteBio for the Octet sample and reagent plate.

 

ITC Resources

CMI ITC200 Getting Started Guide, general guidelines for sample preparation and standard protocol

MicroCal ITC200 Getting Started Handbook
MicroCal ITC200 User Manual

Supplies provided by the CMI:

  • tubes for syringe filling
  • water and methanol for instrument cleaning

Assay Buffers

  • 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.
  • DMSO has high heats of dilution and should be matched extremely well between the cell and the syringe.
  • Small differences in pH will cause high heats of dilution.
  • Reducing agents can cause erratic baseline drift and artifacts. TCEP is recommended over βMe and DTT. Avoid or keep ≤ 1 mM, especially if ΔH is small.
  • Using degassed buffers will reduce the introduction of air bubbles and improve results.

Samples

  • Required volumes for one experiment:
    • ≥ 300 µL protein for sample cell (202 µL + ~ 80-90 µL for filling).
    • ≥ 100-120 µL ligand for syringe (40 µl syringe + 20 µl for filling, for each injection)
  • typical starting concentrations:
    • 5-50 µM in the cell (at least 10x Kd) 
    • 50-500 µM in the syringe (≥10x concentration in cell for 1:1 stoichiometry
      • Aim for a c value between 10-100 for optimal fit  
  • Molar concentration should be accurately measured.
    • Errors in Cell concentration affect stoichiometry.
    • Errors in the Syringe concentration will directly translate to errors in the KD, and affect ΔH and n.
  • 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.

MST Resources

Required Supplies

  • fluorescent target sample and non-fluorescent ligand sample and matched buffer
  • MST capillaries (see below)
  • 0.2 ml tubes for sample preparation (provided by CMI)
  • pipetors and tips for liquid handing

 

NanoTemper Supplies

MST Capillaries

  • Monolith NT.115 Standard Treated Capillaries, MO-K002 (For now, the CMI has Standard capillaries for use.)
  • Monolith NT.115 MST Premium Coated Capillaries, MO-K025

 Labeling Kits (optional)

  • NanoTemper Protein Labeling Kit RED-NHS 2nd Generation (Amine Reactive), MO-L011
  • NanoTemper Protein Labeling Kit RED-MALEIMIDE 2nd Generation (Cys Reactive). MO-L014
  • NanoTemper His-Tag Labeling Kit RED-tris-NTA 2nd Generation, MO-L018

 

Assay Buffers

  • Many buffers are compatible with MST. It’s usually a good idea to start with a buffer system in which your proteins are well behaved.
  • Addition of 0.05% Tween 20 or other detergent is usually required to prevent sticking of proteins to the capillaries.
  • Each capillary should be prepared with identically matched buffer.
  • Assay buffer (with detergent) is used to dilute the fluorescent molecule to 2X.
  • Ligand buffer is used to dilute the ligand and should match the highest concentration of ligand
  • 0.5-1 mg/ml BSA can also be used to minimize non-specific binding.
  • Buffer cannot be opaque.
  • High viscosity samples may be hard to fill (up to 10% glycerol is fine).

Samples

  • All MST experiments are setup with one fluorescently-labeled molecule (the Target) at a fixed concentration mixed with various concentrations of a non-fluorescent molecule (the Ligand).
  • Concentration should be accurately measured
    • Errors in Target concentration can affect fluorescent signal and may affect the fit
    • Errors in the Ligand concentration will directly translate to errors in the KD
  • Protein aggregates will interfere with MST
    • Filter or centrifuge samples before use.
    • Assess protein heterogeneity via light scattering.
    • Purify protein samples with soluble aggregates by size-exclusion chromatography.

Target Sample (the fluorescent molecule)

  •  ~200 µL/titration at >2X working concentration
  • 5-20 µM unlabeled protein, if using a chemical labeling kit
  • RED detector:
    • Stock concentration of labeled Target: > 10 nM
    • Recommended working concentration: 5 nM (for KD > nM)
    • Minimal working concentration: ≥ 50 pM (used for KD in pM range)
  •  BLUE detector:
    • Stock concentration of labeled Target: > 20 nM
    • Recommended working concentration: 20 nM
    • Minimal working concentration: ≥ 5 nM

Ligand Sample (the non-fluorescent binding partner)

  • ~ 20 µL/titration, at 2X working concentration (bring the highest stock concentration available for an unknown KD)
  • Recommended stock concentration ≥ 100X the expected KD
  • Recommended working concentration ≥ 50X KD

CD Resources

CMI Jasco J-815 CD Getting Started Guide, guide to experimental design and standard Far-UV CD protocols.

 

Protein Circular Dichroism Data Bank, a database of CD data

Dichroweb, an online circular dichroism analysis suite

  

The J-815 User Guide is available at the instrument, but not electronically.

  • High Quality Quartz cuvette
    • Far-UV measurements (protein secondary structure):  1 mm pathlength cuvette (eg. Hellma 110-1-40, style 110-QS, available from Sigma-Aldrich)
    • Near-UV measurements:  1 cm pathlength cuvette (eg. Hellma 100-10-40, style 100-QS, available from Sigma-Aldrich)
  • your sample
  • buffer blank

Assay Buffers

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.
  • Always take a scan of your buffer blank to determine whether absorbance interferes with your region of analysis.
    • Ensure that the buffer blank is well matched to the final dilution of your protein (even trace amounts of some solvents will interfere with the CD measurements).
  • Phosphate buffers with little-to-no NaCl are recommended. 10 mM phosphate buffer is ideal.
  • Ideal aqueous solutions contain as little chloride as possible.
    • NaCl and Tris buffers are not recommended but can be tolerated at low concentrations.
    • If salt is required, SO42- or F- are preferred counter ions, as they are transparent in the far UV. Potassium fluoride may be a good substitute for chloride-containing salts.
    • Chloride ions will result in a loss of signal below about 200±5 nm, which affects secondary structure estimation, but generally will not block detection of the alpha-helical peaks (208 nm and 222 nm) or the beta-sheet peak (218 nm).
  • DTT, ß-ME, or EDTA can be present at low concentrations (1 mM).
  • Some detergents are fairly transparent in the far UV (e.g. Chaps and octylglucoside). Triton detergents should be avoided, as they can oxidize readily and form UV-absorbing materials.
  • DMSO, formamides, and imidazole absorb strongly in the far UV region, and thus should be avoided in a CD experiment.

Samples

  • An accurate protein concentration is required for all CD experiments.
    • Over-estimating or under-estimating the protein concentration interferes with signal
    • Algorithms that estimate secondary structure depend on correct protein concentrations
  • The amount of sample required for a CD experiment depends on the size, cuvette pathlength 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 patch 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.
    • 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.

 

DSF Resources

All Experiments:

  • 96-well FAST-block optical plate, eg.: LifeTechnologies MicroAmp FAST optical 96-well reaction plate, 0.1 ml, 4346907
  • optical adhesive film, eg.: LifeTechnologies MicroAmp Optical Adhesive Film, 4360954

DSF/Protein Thermal Shift Experiments

  • DSF compatible dye, eg.: LifeTechnologies Protein Thermal Shift Dye Kit, 4461146 (Sypro Orange)
  • samples, ligands, buffers 

qPCR Experiments

  • qPCR reagents (eg. LifeTechnologies PowerUp SYBR Green Master Mix, A25742)
  • primers and templates 

qPCR Resources

Light Scattering Resources

DLS Resources

CMI DynaPro DLS Getting Started Guide, guide to DynaPro Plate Reader III.

Tips for evaluating correlation function, adapted from DYNAMICS User’s Guide (M1406 Rev. F).

DLS Technology page from Wyatt Technologies.

DLS Supplies

0.02  μm filters for sample preparation, eg. Whatman Anatop 10, 0.02 μm, 10 mm inner diameter, Part Number 6809-1002

Clear-bottom Black Microplates:

number of wells 384 384 96
Manufacturer Aurora Microplates Greiner Bio-One Greiner Bio-One
Part Number ABM2‐10100A 781 892 655 892
base material 188 μm film glass glass
Minumum Volume 25 μl 50 μl 100 μl
Temperature Range 20-85 C 20-37 C 20-37 C
SLS Compatible yes yes yes
Recommended For high temperature/
thermal stability
Room Temp Room Temp

 

 

Generally, SEC-MALS data collection is done in ASTRA 7 with HPLC control.  

CMI SEC-MALS Getting Started Guide

CMI SEC-MALS Guide to Protein Conjugate Analysis, guide to measuring dn/dc values and performing protein conjugate data analysis.

CMI SEC-MALS Guide to Working with Custom Solvents, for solvents that cannot use PBS or water as the solvent model.

SEC-MALS Technology page from Wyatt Technologies.

 

  • purified protein samples (must be filtered before loading)
  • running buffer (1L)
  • analytical SEC column (the CMI usually has 1-2 working SEC-MALS columns that users may borrow, if their samples are purified by SEC and in an appropriate buffer)

 

Assay Buffers

  • Running buffer should always be chosen to be compatible with both the SEC column and the protein sample.
  • Recommended Buffer: 25 mM HEPES pH 7-7.5, 150 mM NaCl (filtered).
  • Make sure you know the buffer compatibility of the SEC column you are using.
    • Most silica columns will not tolerate pH above 7.5.
  • Some buffer components (e.g. glycerol) will require customization of the solvent profile. Take note of the absolute refractive index of the solvent after equilibration and before data collection.

Samples

  • The sample should be prepared in running buffer to minimize RI peak due to sample solvent.
    • This is particularly important for samples that run near the solvent peak.
    • It is good practice to run a size-exclusion chromatography purification prior to analytical SEC-MALS to buffer exchange and clear aggregates. This is a requirement if you are using a shared CMI column.
  • Samples must be filtered or centrifuged prior to injection.
  • Concentration should be accurately measured to assess column recovery.
    • Know the concentration (in mg/ml) of your protein.
    • Know the UV extinction coefficient (in ml/mg•cm) of your protein
  • Protein aggregates can damage the column.
    • Filter or centrifuge samples before use.
    • Assess protein heterogeneity via dynamic light scattering (in the DynaPro plate reader).
    • Purify protein samples with soluble aggregates by size-exclusion chromatography.
  • Recommended protein concentration varies depending on protein mass:
    • Scattering is proportional to mass
      • Larger proteins require less sample than smaller proteins.
    • Typical range 5 – 500 µg/injection
      • BSA (67 KDa) 100 µl at 2 mg/ml always gives a good light scattering signal
  • Sample volume:
    • Maximum injection volume: 100 µl
    • Minimum injection volume: 5 µl
    • Glass autosampler vial with low volume insert has 10 µl dead volume.
      • Fill vial with at least 110 µl for a 100 µl injection.

 

 

Assay Buffers

  • DLS is compatible with a range of buffers
  • Use filtered buffers
  • Measure scattering of buffer control
    • Detergents frequently have micelle size similar to that of proteins and scatter significantly

Samples

  • DLS is very sensitive to small amounts of aggregates.
    • Significant aggregates will make accurate size measurements difficult.
    • Samples should be filtered or centrifuged (6000g, 10-30 min) prior to transfer to the clear bottom plate to remove large aggregates and precipitates.
  • The minimum amount of sample required depends on the size of the molecule, as large molecules scatter more light than small molecules.
    • For most protein samples, concentrations of 0.2 mg/ml or higher are recommended.
    • As little as 0.125 mg/ml of lysozyme or 12.5 μg/ml of IgG can be detected.
  • Small molecule aggregation analysis should be done at working concentrations in and working solvents (e.g. 5% DMSO).
    • Solvent controls are essential. DMSO itself is prone to aggregation.
  • When working with membrane proteins, note that empty detergent micelles will not resolve from protein-filled micelles (except when empty micelle varies in size by 5-10X)
    • Avoid excess detergent by purifying membrane proteins by affinity and/or size-exclusion chromatography and avoid concentrating after purification