Tetra Detector Analysis of Membrane Proteins

Larry J.W. Miercke1, Rebecca A. Robbins1, Robert M. Stroud1

1 Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 29.10
DOI:  10.1002/0471140864.ps2910s77
Online Posting Date:  August, 2014
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Well‐characterized membrane protein detergent complexes (PDC) that are pure, homogenous, and stable, with minimized excess detergent micelles, are essential for functional assays and crystallization studies. Procedural steps to measure the mass, size, shape, homogeneity, and molecular composition of PDCs and their host detergent micelles using size‐exclusion chromatography (SEC) with a Viscotek Tetra Detector Array (TDA; absorbance, refractive index, light scattering, and viscosity detectors) are presented in this unit. The value of starting with a quality PDC sample, the precision and accuracy of the results, and the use of a digital benchtop refractometer are emphasized. An alternate and simplified purification and characterization approach using SEC with dual absorbance and refractive index detectors to optimize detergent and lipid concentration while measuring the PDC homogeneity is also described. Applications relative to purification and characterization goals are illustrated as well. Curr. Protoc. Protein Sci. 77:29.10.1‐29.10.30. © 2014 by John Wiley & Sons, Inc.

Keywords: membrane proteins; tetra detector array and analysis; differential pressure viscometer; intrinsic viscosity; refractive index

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Table of Contents

  • Introduction
  • Basic Protocol 1: Tetra Detector (Absorbance, Refractive Index, Light Scattering, and Viscosity) Analysis for Complete Characterization of Purified PDC and Host Detergent Micelle
  • Alternate Protocol 1: Dual‐Absorbance and Refractive Index Detection to Enhance PDC Homogeneity Measurements, Optimize Detergent Concentration, Define SEC Purification Limitations, and Ensure Sample Quality for Tetra Detection
  • Support Protocol 1: Using a Digital Bench‐Top Refractometer to Measure Molecular dn/dc and Refractive Index of SEC Column Buffer (RISOL)
  • Support Protocol 2: Purification of Ovalbumin Factor VII for Measuring TDA Detector Response Factors
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Tetra Detector (Absorbance, Refractive Index, Light Scattering, and Viscosity) Analysis for Complete Characterization of Purified PDC and Host Detergent Micelle

  • SEC column buffer (see recipe)
  • TDA standard (see recipe)
  • Detergent stock in column buffer (see recipe)
  • Sample: quality PDC (pure, homogenous and stable with minimal detergent) in column buffer at > 0.6 mg/ml (see recipe)
  • 0.025% sodium azide in water, or other bacteriostatic reagent, e.g., 20% ethanol.
  • SEC column: identical to that used to produce and support a homogenous and stable (uniform population of molecules that do not change over time) PDC. Recommended columns are the silica‐based TSK G3000SW 0.75 × 60 cm with a 0.75 × 5 cm guard column (10 µm particle size; 250 Å pores; 735 maximum psi; 2.5‐7.5 pH range), and the linked agarose/dextran‐based Superdex (SDX) 200 10/300 (1 × 30 cm; 13 µm average particles size, unspecified pore sizes, 218 maximum psi; 3 to 12 pH range) without a guard column. The TSK column was used for this protocol 1Basic Protocol.
    • To inhibit matrix compression and band broadening, the manufacturer's recommended maximum pressure across the column (post sample injector to post detectors) must not be exceeded. Column pressure is dependent on column chemistry, solvent viscosity, tubing diameter, and on the number of detectors and their flow cell size and geometry. The final TDA flow rates tend to be just below the column maximum, and usually range from 0.3 to 0.6 ml/min. Even though a replaceable filter is installed by the manufacturer in front of the LS detector flow cell, a new column should not be used in order to further minimize any possible effects of column shedding.
  • Tetra Detection Work Station: composed of:
    • A stable isocratic pump with piston backflush and non‐leaking seals and pistons
    • Four properly maintained detectors (absorption, differential refractometer, static light scattering and absolute differential pressure viscometer)
    • Calibrated syringe or auto sampler for accurate sample volume injections
    • An on‐line degasser to ensure stable signals with minimal spikes and noise
    • A computer interface for data collection and analysis
    • Tubing used to connect the pump and injector to the tetra detector array should be short and of small diameter to minimize band broadening
    • To ensure a constant temperature for accurate and reproducible results (usually 4°C), all components except the degasser (which generally will not work at cold temperatures due to incompatible seals) and computer are placed in a temperature‐controlled chromatography room or box.
    • All our direct experiences and presented data used a Viscotek system composed of a VE 1122 dual piston isocratic pump, manual 7725i Rheodyne sample injector valve, 500 µl PEEK sample loading loop, blue (0.01 in. ID × 1/16 in. OD) PEEK tubing, and a tetra detector array (model 302 triple array and Knauer K‐2501 absorbance detector), all residing in a Puffer Hubbard dedicated 4°C chromatography refrigerator.
    • The VE 7510 GPC degasser (Viscotek) and PC computer running Windows both reside outside the refrigerated box at room temperature. To accommodate the degasser, the solvent resides at room temperature, and is connected to the degasser using 0.093 in. ID × 1/8 in. OD tubing and 10‐µm stainless steel solvent inlet stone; a coiled 10 foot piece of 0.25 mm diameter ID stainless steel tubing placed in the refrigerator and between the de‐gasser and pump ensures the solvent is temperature equilibrated before it enters the pump. Using this configuration, the temperature at the LS detector cell is a constant 6°C (temperature probe located at LS flow cell). A 100‐, 200‐, or 500‐µl Hamilton gas‐tight syringe, calibrated using water and an analytical balance, is used for sample injections. This absolute viscometer measures DP between eluting peak and solvent by splitting the incoming SEC liquid stream and delaying one stream with an internal size exclusion delay column, and then comparing the output pressure of each side of the stream. With this design, the DP profile for each eluting component consists of a positive peak followed by a delayed negative peak.
    • An on‐line degasser may not be required when properly degassed column buffer is used, and a calibrated auto‐sampler will minimize total time required to complete a TDA experiment. Samples and syringes are always kept on ice or in the 4°C chromatography refrigerator.
  • Viscotek's OmniSEC software version 4.1 running on a Windows‐based PC computer is used for data collection and analysis. Every tetra detector chromatogram (TDAgram) has a data file with associated Run Parameters (sample name, date and time, injection volume, sample concentration and column flow rate). Using the Run Parameters and a created Method file, the TDAgram is calibrated to establish calibration response factors and Executed to output results using defined integration limits and baselines. Each Method file includes a defined Method Type; for a single‐component molecule like a soluble protein or detergent micelle, the Multi‐detector homopolymer method type (mdHP) is utilized for data analysis, while the Multi‐detector Copolymer (mdCP) method type is used to analyze a two‐component, single‐scattering particle such as a PDC (Fig.  ). Other method types within the OmniSEC data analysis module are available and useful depending on detectors used and knowledge of molecules under study. Even though only one mdHP method type is used to analyze each single‐component TDAgram, for clarity, four different sequential iterations are described and termed Individual mdHP K Method, Average mdHP K Method, RI mdHP Area Method, and Micelle mdHP Method. The final PDC mdCP Method incorporates the mdCP method type and uses concentration data from the UV as well as the RI detectors for the final analysis of the PDC. Microsoft Excel and GraphPad Prism (or any similar spreadsheet programs) can be used to assist in data analysis and result summaries. For convenience, during data collection, a second Windows‐based PC running OmniSEC software is used for data analysis.
    • Equations used for accumulated statistics for each data set are (1) relative error or error in average = SD/sqrt N, where SD and N represents the standard deviation and number of measurements, respectively, (2) absolute error or % error in average = (relative error/average) × 100, and (3) % average error = ((average‐predicted)/predicted) × 100.
    • To properly use all capabilities of the extensive OmniSEC software, it is advised to study and understand the Method types, equations used, input parameters and output results before experiments are considered and planned.

Alternate Protocol 1: Dual‐Absorbance and Refractive Index Detection to Enhance PDC Homogeneity Measurements, Optimize Detergent Concentration, Define SEC Purification Limitations, and Ensure Sample Quality for Tetra Detection

  Additional Materials (also see protocol 1Basic Protocol)
  • PDC sample in SEC running buffer as described in the protocol 1Basic Protocol materials list, but the PDC does not necessarily need to be pure, homogenous, and stable
  • Equipment is an isocratic chromatography workstation composed of a pump, sample injector, absorbance and RI detectors, fraction collector, and computer software. The authors currently use a Shimadzu binary workstation composed of an SCL‐10Avp system controller, two LC‐10ADvp pumps, SPD‐M20A diode array detector, RID‐10A refractive index detector, SIL‐10AP automatic sample injector, FRC‐10A fraction collector, Windows‐based computer, and Class‐VP 7.4 software.

Support Protocol 1: Using a Digital Bench‐Top Refractometer to Measure Molecular dn/dc and Refractive Index of SEC Column Buffer (RISOL)

  Additional Materials (also see protocol 1Basic Protocol)
  • A properly calibrated bench‐top refractometer is required. The refractometer should have a digital processor with digital outputs to at least five decimal places (Straat and Forrest, ), stable temperature control, and possible variable wavelengths (RI is dependent on both temperature and wavelength). An analog Abbe‐3L refractometer (Fisher Scientific) placed at 4°C, originally used, was found to have inadequate resolution and accuracy for RI analysis. But a single‐wavelength (λ = 589.29 nm; considered standard) Anton Paar Abbemat RXA156 digital refractometer (now called Abbemat HP, High Precision) and Windows‐based PC computer is precise and accurate to 6 decimal places with fast Peltier temperature changes (10° to 70°C range; 2‐min equilibration for 2° changes), and a 150‐µl sample cell (50 µl sample cell available) (Fig.  ). Calibration is performed using water and can be easily corrected for small differences in wavelength and temperature if desired (Tilton and Taylor, ).

Support Protocol 2: Purification of Ovalbumin Factor VII for Measuring TDA Detector Response Factors

  • Ovalbumin Fraction VII (Sigma, cat. no. A7641‐1g)
  • SEC column buffer (see recipe)
  • Analytical balance
  • Temperature‐controlled microcentrifuge (e.g., Eppendorf 5415 R)
  • 500‐µl Hamilton gas‐tight syringe (Fig.  )
  • SEC column identical to that used for TDA ( protocol 1Basic Protocol)
  • Isocratic SEC station with absorbance detector, sample injector, and fraction collector. The authors currently use a Shimadzu SCL‐10Avp system controller, LC‐20AD pump, SPD‐20A dual‐wavelength UV/Vis detector, FRC‐10A fraction collector, manual 7725i Rheodyne sample injector valve, and 0.5 ml PEEK sample loading loop.
  • Spectrophotometer with a good optical bench. A Shimadzu 2501‐PC UV‐Vis or Agilent 8453 UV‐visible diode array is utilized with a quartz, masked, and 10‐mm‐path cuvette.
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Internet Resources
  Web sites for information on the technology, techniques, schematics, instrument care, and applications for tetra detector arrays and analysis.
  Web site for information on bench‐top refractometers.
  Web sites for information on TSK, SDX, SRT, and Nanofilm SEC columns.
  Web sites for information on detergents and lipids.
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