93Methods in Cell Biology, Vol 112 Copyright © 2012 Elsevier Inc. All rights reserved.

0091-679Xhttp://dx.doi.org/10.1016/B978-0-12-405914-6.00005-6

Mads Gabrielsen*, Katherine S. H. Beckham, Andrew J. RoeInstitute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life

Sciences, University of Glasgow, Glasgow, UK

*Corresponding author: mads.gabrielsen@glasgow.ac.uk

CHAPTER

High-Throughput Methods for the Identification of Protein Purification Conditions Using a Cleavable Tag System

5

CHAPTER OUTLINE

1 Purpose ................................................................................................................. 942 Theory ................................................................................................................... 943 Equipment ............................................................................................................. 954 Materials ............................................................................................................... 95

4.1  Solutions & Buffers ...............................................................................964.2  Solutions & Buffers—Step 1 ..................................................................964.3  Solutions & Buffers—Step 2 ..................................................................974.4  Solutions & Buffers—Step 3 ..................................................................974.5  Solutions & Buffers—Step 4 ..................................................................98

5 Protocol ................................................................................................................ 985.1  Step 1—Membrane preparation .............................................................985.2  Step 2—Membrane solubilization .........................................................1005.3  Step 3—Affinity chromatography ..........................................................1005.4  Step 4—Desalting of the proteins .........................................................1035.5  Step 5—TEV cleavage .........................................................................1055.6  Step 6—Second affinity chromatography step .......................................1065.7  Step 7—Protein quantitation ...............................................................107

AbstractStructural and biochemical characterization of integral membrane proteins relies on developing appropriate purification protocols for the individual protein. This step often acts as a bottleneck, as the purification process is complicated by the need for

CHAPTER 5 High-Throughput Methods94

detergents to remove the membrane protein from its lipid environment, and to main-tain them in solution throughout the experiment. The choice of detergent can have a significant effect on the protein as the formation of the protein–detergent complex is dependent on the occurrence of favorable interactions between the protein and the detergent. These interactions cannot be predicted, and require empirical testing. This protocol describes a high-throughput method to determine the best purification conditions for several proteins simultaneously. Here we describe a protocol to test the purification of 12 membrane proteins using the eight most commonly used detergents (in X-ray crystallography). The protocol relies on the use of a construct encoding a His-tagged fluorophore and a His-tagged protease.

1 PURPOSEThe purpose of this protocol is to identify the best purification detergents for 12 inner membrane proteins using high-throughput methods, requiring small amounts of sample.

2 THEORYPurification of integral membrane proteins (IMP) is becoming standard in many labs. However, this process carries with it some particular difficulties, mainly the need for detergents to remove the IMP from their natural hydrophobic environment, and maintaining them in solution. The disruption of the cell membrane releases the IMP into a protein–detergent complex (PDC). Detergents have to be present throughout the purification process, at a concentration above the critical micelle concentration (CMC) (for details see review by Privé, 2007). The resulting PDC has its own bio-physical properties dependent on both the protein and the detergent. These properties cannot be predicted, and thus need to be empirically tested. As there is a wide range of detergents available a high throughput approach to the testing of detergent suit-ability is preferable.

This protocol details the use of high-throughput methods to test the most suited purification detergents for a number of IMP, simultaneously.

Using a 96-well format (12 × 8), 12 IMP (Columns C1-C12) can be tested in eight different detergents (Rows D1–D8), using Nickel affinity chromatography. The cell membranes are initially solubilized in n-Dodecyl-β-d-maltopyranoside (DDM), as this is commonly used, and reasonably inexpensive (McLuskey et al., 2008). Follow-ing initial solubilization, the IMP exchanged into the eight different detergents, which takes place on the Ni2+ columns.

Utilizing fluorescent markers, such as GFP (Green fluorescent protein), to monitor membrane protein purification, and to identify the best initial purification detergent, has been used with some success (Gabrielsen et al., 2011). This protocol relies on the use of a construct that encodes a His-tagged GFP fused to the protein of interest. Further, a protease cleavage site needs to be incorporated between GFP and the IMP.

3 Equipment 95

A His-tagged version of a protease is also required to allow an easy two-step purification. This protocol describes the use of TEV protease, but this can easily be replaced with 3C protease, if preferred.

The purification is based on Immobilized Metal Affinity Chromatography, where solubilized membranes are loaded onto Ni2+ columns, washed and eluted. The eluted protein is desalted, and mixed with TEV protease to cleave off the GFP–His–tag. As the TEV protease is also His-tagged, a secondary Ni2+ column will bind the cleaved GFP-His-tag and the TEV protease, and the membrane protein, which will not bind, is collected in the in the flow-through.

The detergents chosen by the authors are based on the most successful crystalliza-tion experiments published in the Protein Data Bank, and are listed below. However, these can be replaced depending on the experimenter’s preferences. The appropriate concentration of detergent to reach the CMC has been calculated for the detergents used in this study, and will need to be altered for any different detergents that the user may have introduced. Similarly, other fluorophores may be used instead of GFP in which case the excitation and emission wavelengths may need to be altered to detect the presence of protein. The user should refer to the published works on the specific fluorophores for details.

3 EQUIPMENTFalcon tubes (50 ml)CentrifugeUltracentrifugeUltracentrifuge tubesQIAFilter plate, QIAGENPhotospectrometer (Absorbance at 280 nm, A

280)

Platereader Fluorimeter, with excitation/emission wavelengths of 485/520 nm, e.g. FLUOstar OPTIMA, BMG Labtech96-well Deep well block (DWB, AbGene, Thermo Scientific)96-well analysis plates (Corning Ltd)Centrifuge with rotor suitable for Deep well blocks (such as Beckman Allegra X-12R, with an SX4750 rotor).

4 MATERIALSNiNTA Superflow (QIAGEN)Desalting block (Zeba, PIERCE)MgCl

2

NaClTris–HClImidazoleDNAse

CHAPTER 5 High-Throughput Methods96

Complete Inhibitor (EDTA free) cocktail (Roche)His-tagged TEV protease

Detergents listed below

4.1 Solutions & Buffers

Component

Final Concentration in Buffers Stock

Amount for Buffers

D1 n-Dodecyl-β-D-maltopyranoside DDM 0.04 10% 20 µLD2 n-Decyl-β-D-maltopyranoside DM 0.20 10% 100 µLD3 n-Octyl-b-D-glucopyranoside β-OG 1.00 10% 500 µLD4 n-Nonyl-b- D-glucopyranoside NG 0.40 10% 200 µLD5 N-Dodecyl-N-N-

dimethylamine-N-oxideLDAO 0.10 10% 50 µL

D6 CYMAL-5 CY-5 0.30 10% 150 µLD7 Fos-choline-14 FC-14 0.04 10% 20 µLD8 ANAPOE-C12E8 C12E8 0.01 10% 5 µLTip Make sure the detergent powder is crushed into a fine powder before added to the 

solution. Make up in MB (see below).

Stock solutions Stock Amount

Tris–HCl 1 M 157.6 gMake up 800 mL in distilled H2O, and pH to 7.5, using NaOH. Make to a final volume of 1 L using distilled H2O.NaCl 4 M 58.44 gMake up 1 L in distilled H2O.Imidazole pH 7.5 1 M 34.04 gMake up 500 mL in distilled H2O and pH to 7.5, using HCl. Make up to a final volume of 1 L, using distilled H2O.EDTA 0.1 M 0.372 gMake up 10 mL in distilled H2O.DTT 0.1 M 0.154 gMake up 10 mL in distilled H2O.

4.2 Solutions & Buffers—Step 1

Component Final concentration Stock Amount

Lysis Buffer, LYB

Tris–HCl pH 7.5 50 mM 1 M 50 ml

MgCl2 1 mM 95.21 mg

4 Materials 97

Component Final concentration Stock Amount

DNAse 50 U/ml 552 U/mg 90 mgComplete EDTA-free protease inhibitor

1 tablet

Make up Tris–HCl and MgCl2 in 1 L distilled H2O. Add DNAse and dissolve protease inhibitor tablet.

Storage Buffer, SB

Tris–HCl pH 7.5 20 mM 1 M 2 mlNaCl 50 mM 4 M 1.25 mlGlycerol 10% 100% 10 mlMake up final volume of 100 ml.

4.3 Solutions & Buffers—Step 2

Component Final concentration Stock Amount

Membrane Buffer, MB

Tris–HCl pH 7.5 20 mM 1 M 2 mlNaCl 50 mM 4 M 1.25 mlMake up final volume of 100 ml.

4.4 Solutions & Buffers—Step 3

Component Final concentration Stock Amount

Membrane Buffer with Detergent, MBD1-8

Tris–HCl pH 7.5 20 mM 1 M 100 µLNaCl 50 mM 4 M 62.5 µLDetergent See table aboveMake up 5 ml of each MBD buffer. Dissolve detergents (1–8, see table) in the volume.

Wash Buffer with Detergents, WBD1-8

Tris–HCl pH 7.5 20 mM 1 M 100 µLNaCl 50 mM 4 M 62.5 µLImidazole 50 mM 1 M 250 µLDetergent See table aboveMake up 5 ml of each WBD buffer.

Elution Buffer with Detergents, EBD1-8

Tris–HCl pH 7.5 20 mM 1 M 100 µLNaCl 50 mM 4 M 62.5 µLImidazole 250 mM 1 M 250 µLDetergent See table aboveMake up 5 ml of each EBD buffer.

CHAPTER 5 High-Throughput Methods98

4.5 Solutions & Buffers—Step 4

Component Final concentration Stock Amount

Desalting buffer with detergents, DBD1-8

Tris–HCl pH 7.5 20 mM 1 M 100 µLNaCl 50 mM 4 M 62.5 µLEDTA 0.5 mM 0.1 M 25 µLDTT 10 mM 0.1 M 500 µLDetergent See table aboveMake up 5 ml of each EBD buffer.

5 PROTOCOL

Duration Time

Preparation 1 dayProtocol 4 days

Preparation Grow twelve 500 ml cultures of overexpressed inner membrane proteins and store membranes at −80 °C until required (details for membrane preparation described below).Prepare buffer blocks by using 96 DWB (2 ml) with D1 in Row A, D2 in Row B, and so forth (Fig. 2). Two buffer blocks are required for buffers MBD1-8, and one of each of buffers WBD1-8, EBD1-8 and DBD1-8.

CautionTip Step 1 is done before the experiment starts, and does not need to be 

done on all 12 samples at once.A schematic is presented in Fig. 1 to illustrate the workflow of the experiment Fig. 2.

5.1 Step 1—Membrane preparation

Overview Lyse the cells and prepare the membranes. Store at −80 °C until ready for experiment.

Duration 2 h1.1 Thaw cell pellet and resolubilize in 30–50 ml LYB.1.2 Lyse the cells using mechanical pressure (e.g. French press or a cell 

disruptor).1.3 Transfer cell lysate to 50 ml Falcon tubes.1.4 Centrifuge lysate at 9000g for 30 min at 4 °C.1.5 Decant supernatant into ultracentrifuge tubes.1.6 Harvest the membranes by centrifugation at 100,000g for 1 h at 4 °C.1.7 Discard the supernatant. Carefully resuspend the membranes in SB, 

using a small syringe with a needle (21 gauge).1.8 Transfer to prelabeled Eppendorf tubes.1.9 Flash freeze in liquid nitrogen and store at −80 °C.

5 Protocol 99

Preparation

Step 1

Step 2

Step 3

Step 4

Step 5

Over-express 12 membrane proteins

Membrane preparation

Membrane solubilisation

Desalt protein-detergent samples

TEV cleavage

Primary Nickel affinity chromatography

Step 6 Secondary Nickel affinity chromatography

Step 7 Protein quantitation

FIGURE 1

Flowchart of the complete protocol, including preparation steps.

FIGURE 2

96-well Deep well block, used throughout protocol. Each row contains one detergent (D1–D8) and each column contains one membrane protein (P1–P12).

CHAPTER 5 High-Throughput Methods100

Caution The membranes should be as homogenized as possible. Repeated aspiration and dispersion with the syringe should be adequate. Avoid frothing the membranes.

Tip The insoluble fractions can be frozen and kept for analysis, if they appear green, or if there is a very low amount of protein in the solubilized membranes. Although the authors prefer mechanical pressure to lyse the cells, other alternatives are routinely used in other labs (e.g. sonication).

See Fig. 3 for the flowchart of Step 1.

5.2 Step 2—Membrane solubilization

Overview Prepare the membranes for the HTP purification experiment.Duration 2.5 h2.1 Thaw all twelve-membrane preparations on ice (P1–P12)2.2 Dilute to 10 ml in MB.2.3 Measure the absorbance at 280 nm (A280) to determine the protein 

concentration of each sample. Assume an extinction coefficient of 1 for the mixed population, and apply Beer–Lamberts law (c = A280 × MW, assuming a path length of 1)

2.4 Dilute all samples to approximately 30 mg/ml.2.5 Add powdered DDM stepwise to each membrane sample to reach a final 

concentration of 1.5%. Ensure the detergent has been crushed into a fine powder.

2.6 Make note of any clearing of the solution occurring when the detergent is added.

2.7 Incubate samples on ice for 1 h.2.8 Pour samples into ultracentrifuge tubes and centrifuge at 100,000g for 

45 min at 4 °C.Tip In the authors’ experience, an immediate clearing of the membrane 

solution is often observed in successful purification preps. Any cloudy solutions after 1 h are less likely to yield large amounts of protein.

See Fig. 4 for the flowchart of Step 2.

5.3 Step 3—Affinity chromatography

Overview HTP affinity purification of 12 membrane proteins in eight different detergents.

Duration 6 h3.1 Place a QIAFilter plate on top of an empty 96 DWB.3.2 Prepare 96 Ni2+ columns by loading 500 µL Ni-NTA. suspension onto a 

QIAFilter plate (leaving a column volume of 250 µL).3.3 Equilibrate QIAFilter plate in 750 µL MBD1-8, with Buffer MBD1 in row 1, 

MBD2 in row 2 and so forth. Discard all flow-through.3.4 Split each solubilized membrane protein sample into eight equal aliquots.3.5 Load 750 µL of P1–P12 onto columns 1–12 of the plate, leaving one 

protein per column (as per Fig. 2).

5 Protocol 101

Step 1 : Membrane preparation

1.1 Thaw cell pellet and resolubilise in LYB.

1.2 Lyse cells.

1.3 Transfer to 50 ml Falcon tubes.

1.4 Centrifuge at 9,000g for 30 min at 4°C.

1.5 Decant supernatant into ultracentrifuge tubes.

1.6 Harvest membranes by ultracentrifugationat100,000g for 1 hour at 4°C.

1.7 Discard supernatant. Resuspend the pellet in SBwith a small syringe and needle.

1.8 Transfer to pre-labelled Eppdendorf tubes.

1.9 Flash freeze in liquid nitrogen and store at –80°C.

FIGURE 3

Flowchart of step 1.

CHAPTER 5 High-Throughput Methods102

Step 2 : Membrane solubilisation

2.1 Thaw all twelve membranepreparations on ice (P1-P12).

2.2 Dilute to 10 ml in MB.

2.3 Determine concentration.

2.4 Dilute all samples to approx. 30 mg ml-1.

2.5 Add powered DDM to a final concentration of 1.5 % (w/v).

2.6 Watch for clearing of the solution.

2.7 Incubate samples on ice for 1 h.

2.8 Decant samples into ultracentrifuge tube and centrifuge at100,000g for 45 min at 4°C.

FIGURE 4

Flowchart of step 2.

5 Protocol 103

3.6 Centrifuge at 100g for 15 min. Collect the flow-through in a 96-well DWB.3.7 Repeat 3.5 and 3.6 until all the samples have been loaded onto the 

columns.3.8 Wash the Ni2+-plate in 600 µL WBD1-8 and centrifuge at 1000g for 1 min. 

Discard the flow through, unless obviously colored.3.9 Repeat 3.8 three times in total.3.10 Collect the elute in fresh DWB, by loading 600 µL of EBD1-8 onto the 

Ni2+-plate, and centrifuge at 5000g for 2 min.3.11 Repeat 3.10 three times, collecting the elutes in three separate blocks.3.12 Determine the level of fluorescence in the three elution blocks, and pool 

them if deemed high enough.Tip Evidence of green in the flow-through when loading the protein samples, 

suggests that the binding capacity of the column in the specific detergent has been reached. At this stage, the fluorescence can be estimated by eye. As much protein as possible should be taken further, so clearly green fractions in elution blocks 2 or 3 should be pooled with 1. However, all samples of one protein needs to be treated the same, whole columns have to be pooled. The eluted samples can be left overnight at 4 °C.

See Fig. 5 for the flowchart of Step 3.

5.4 Step 4—Desalting of the proteins

Overview HTP desalting of 12 proteins in eight different detergents.Duration 1 day4.1 Equilibrate 96 desalting columns (PIERCE) in buffers DBD1-8, by adding 

250 µL into each column and centrifuge at 1000g for 2 min.4.2 Repeat the equilibration three times.4.3 Load 100 µL of the eluted samples onto the desalting plate, maintaining 

the grid system, and centrifuge at 1000g for 2 min. Collect flow through into clean DWB.

4.4 Remove collection DWB, and re-equilibrate the columns as 4.2.4.5 Repeat steps 4.3 and 4.4 until all the samples have been desalted. The 

samples can be collected into the same DWB throughout.4.6 Take 10 µL of sample from each well, using a multichannel pipette. Add 

to an analysis plate, with 90 µL buffers MDB1-8 in the standard grid format.

4.7 Measure absorbance at 280 nm.4.8 Calculate the highest concentration present.Caution The thorough wash of the desalting columns between each load of 

protein is to ensure no transfer of imidazole from this step into the next. Whilst time consuming, the authors have taken a cautious approach and would not recommend cutting down on the number of steps.

See Fig. 6 for the flowchart of Step 4.

CHAPTER 5 High-Throughput Methods104

Step 3 : Affinity chromatography

3.1 Place QiAFILTER on top of empty 96 DWB.

3.2 Prepare 96 Ni 21 clumns by loading 500 µl Ni_NTA suspension onto a QIAFILTER plate.

3.3 Equilibrate QIAFILTER platein 750 µl of buffer.

3.4 Divide each solubilsed membrane protein fraction into eight equal aliquots.

3.5 Load 750 µl of P1-P12 onto columns 1-12 of the plate, leaving one protein per column.

3.6 Centrifuge at 100g for 15 minutes. Collect the flow-through in a 96 well DWB.

3.7 Repeat 3.5 and 3.6 until all the samples have been loaded onto the columns.

3.8 Wash the Ni 2+ -plate in 600 µl WBD1-8 and centrifuge at 1000g for 1 minute. Discard the flowthrough.

3.9 Repeat 3.8 twice.

3.10 Collect the elute in fresh DWB, by loading 600 µl of EBD1-8 onto the Ni2+ -plate,andcentrifuge at 5000g for 2 minutes.

3.11 Repeat 3.10 three times, collecting the elutes in three separate blocks.

3.12 Determine the level of fluorescence in the threee lution blocks.

FIGURE 5

Flowchart of step 3.

5 Protocol 105

5.5 Step 5—TEV cleavage

Overview Removing the GFP-affinity tag by TEV protease cleavage.Duration 2–3 h or overnight5.1 Add TEV to each sample in a 2:1 ratio (Protein:TEV) based on the highest 

protein concentration calculated.

Step 4 : Desalting the proteins

4.1 Equilibrate 96 desalting columns in buffers DBD1-8 by adding 250µl into each column and centrifuge at 1000g for 2 min.

4.2 Repeat the equilibration three times.

4.3 Load 100 µl of the eluted samples onto the desalting plate, maintaining the gridsystem, and centrifuge at 1000g for 2 min. Collect flow through into clean DWB.

4.4 Remove collection DWB, and re-equilibrate the columns as 4.2.

4.5 Repeat steps 4.3 and 4.4 until all the samples have been desalted. The samplescan be collected into the same DWB throughout.

4.6 Take10 µl of sample from each well, using a multi-channel pipette. Add to ananalysis plate, with 90 µl buffers MDB1-8 in the standard grid format.

4.7 Measure absorbance at 280 nm.

4.8 Calculate the highest concentration present.

FIGURE 6

Flowchart of step 4.

CHAPTER 5 High-Throughput Methods106

5.2 Incubate at 30 °C for 1–2 h, or at 4 °C overnight.5.3 Filter all samples through a 96-well QIAFilter plate to remove any 

precipitation.5.4 Collect into clean DWB.Caution Proteins may start to precipitate at 30 °C. To avoid this, leave overnight  

at 4 °C.

See Fig. 7 for the flowchart of Step 5.

5.6 Step 6—Second affinity chromatography step

Overview Separate cleaved, purified protein from GFP-affinity tag and TEV protease.

Duration 1 h6.1 Prepare a second Ni-column plate as described in 3.1. Equilibrate  

as in 3.2.6.2 Load the filtered samples onto the second Ni-plate.6.3 Collect the Flow-through into a clean DWB.Optional6.4 To assess the efficiency of the cleavage, the beads can be washed in 

EBD1-8 and the fractions can be run on a gel. The bands should be of uniform size and correspond to GFP (27 kDa) and TEV (25 kDa) protease.

See Fig. 8 for the flowchart of Step 6.

Step 5 : TEV cleavage

5.1 Add TEV to each sample in a 2:1 ratio (Protein:TEV).

5.2 Incubate at 30 °C for 1-2 hours, or at 4 °C over night.

5.3 Filter all samples through a 96 well QIAFilter plate to remove anyprecipitation.

5.4 Collect into clean DWB.

FIGURE 7

Flowchart of step 5.

5 Protocol 107

5.7 Step 7—Protein quantitation

Overview The same amount of each protein was loaded onto the eight different detergents. Any differences in recovered protein at the end can therefore be ascribed to the purification detergent used.The amount of protein can be quantified by A280 readings, or, if there are very small amounts left, with the EZQ protein quantitation kit. The authors routinely use both, and present the use of A280 here, as most labs will have routine access to the equipment.

Duration 15 min7.1 Take 100 µL of each sample and transfer to an analysis plate.7.2 Read A280 absorbance in plate reader.7.3 Identify the conditions with the highest protein concentrations for each 

protein, and use these as initial conditions for scale up purification.Optional The rest of the samples can be loaded onto SDS-PAGE Midi gels 

(Invitrogen), to assess purity of the proteins.Caution No comparisons can be made between the different proteins.

See Fig. 9 for the flowchart of Step 7.

Step 6 : Affinity chromatography

6.1 Prepare a second Ni-column plate as described in 3.1.Equilibrate as in 3.2.

6.2 Load the filtered samples onto the second Ni-plate.

6.3 Collect the Flow-through into a clean DWB.

FIGURE 8

Flowchart of step 6.

CHAPTER 5 High-Throughput Methods108

Keywords

Keyword class Keyword Rank Snippet

MethodsList the methods used  to carry out this protocol (i.e., for each step).

1 Membrane preparation

Membranes from E. coli, are separated from the cell lysate by ultracentrifugation.

2 Membrane solubilization

Membrane proteins are released from their membrane environment by solubilization using detergents.

3 Affinity Chromatography

Affinity chromatography relies on an encoded tag (in this case an octahistidine-tag), and a resin that binds the tag (e.g. Ni2+). The non-tagged proteins go through, whereas the tagged protein will bind, and can be eluted at a later stage.

4 Protein desalting Removal of the high salt concentration is essential for the next step.

5 Protein quantitation

Absorbance at 280 nm is a quick and reliable way of determining protein concentration.

Step 7 : Protein quantitation

7.1 Take 100 µl of each sample and transfer to ananalysis plate.

7.2 Read A280 absorbance in plate reader.

7.3 Identify the conditions with the highest concentration for eachprotein, and use these as initial conditions for scale up purification.

FIGURE 9

Flowchart of step 7.

5 Protocol 109

Keyword class Keyword Rank Snippet

ProcessList the biological process(es) addressed  in this protocol.

12345

OrganismsList the primary  organism used in this protocol. List any other applicable organisms.

1 E. coli2345

PathwaysList any signaling, regulatory, or metabolic pathways addressed in this protocol.

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Molecule RolesList any cellular or molecular roles addressed in this protocol.

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Molecule FunctionsList any cellular or molecular functions or activities addressed in this protocol.

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PhenotypeList any developmental or functional phenotypes addressed in this protocol (organismal or cellular level).

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AnatomyList any gross anatomical structures, cellular structures, organelles, or macromolecular complexes pertinent to this protocol.

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DiseasesList any diseases or disease processes addressed in this protocol.

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CHAPTER 5 High-Throughput Methods110

Keyword class Keyword Rank Snippet

OtherList any other miscella-neous keywords that describe this protocol.

1 High-throughput The use of small volumes and 96-well plates allow for rapid testing of a large number of conditions.

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ReferencesSource article(s) used to create this protocolMcLuskey, K., Gabrielsen, M., Kroner, F., Black, I., Cogdell, R. J., & Isaacs, N. W. (2008).

A protocol for high throughput methods for the expression and purification of inner mem-brane proteins. Molecular Membrane Biology, 25, 599–608.

Gabrielsen, M., Kroner, F., Black, I., Isaacs, N. W., Roe, A. J., & McLuskey, K. (2011). High-throughput identification of purification conditions leads to preliminary crystallization conditions for three inner membrane proteins. Molecular Membrane Biology, 28, 445–453.

Referenced literaturePrivé, G. G. (2007). Detergents for the stabilization and crystallization of membrane proteins.

Methods, 41, 388–397.

Related literatureReferenced Protocols in Methods Navigator