Purification of Membrane Proteins Overexpressed in Saccharomyces cerevisiae

Abstract

Membrane protein (MP) functional and structural characterization requires large quantities of high-purity protein for downstream studies. Barriers to MP characterization include ample overexpression, solubilization, and purification of target proteins while maintaining native activity and structure. These barriers can be overcome by utilizing an efficient purification protocol in a high-yield eukaryotic expression system such as Saccharomyces cerevisiae. S. cerevisiae offers improved protein folding and post-translational modifications compared to prokaryotic expression systems. This chapter contains practices used to overcome barriers of solubilization and purification using S. cerevisiae that are broadly applicable to diverse membrane associated, and membrane integrated, protein targets.

1. Introduction

Membrane protein (MP) characterization relies on successful solubilization and purification techniques to generate sufficient yields of purified protein for downstream studies. This chapter provides a set of protocols to overcome common hurdles associated with MP purification, without compromising native protein function and structure. Methods outlined in this chapter are derived from empirical practice and troubleshooting a diverse array of membrane associated and polytopic membrane integrated protein targets. Potential modifications and troubleshooting areas are noted throughout and can maximize purity or recovery for specific proteins of interest (POIs) but protocols are effective for a wide variety of MPs without modification. Content is organized to represent standard workflow of MP expression and purification in S. cerevisiae, starting with growing cultures from transformed colonies to overexpress a POI. After overexpression, POI detergent screens, SDS-PAGE, and immunoblot analysis follows. Finally, POI is purified by immobilized metal affinity columns, size exclusion, and ion exchange chromatography (Fig. 1).

Figure 1: Purification of integral membrane proteins in Saccharomyces cerevisiae Workflow.

Workflow proceeds from top to bottom, between sections and left to right within a section. Five of the primary events for each process are represented as a summary of the techniques covered in this chapter.

2. Materials

All solutions referenced in this chapter are made and stored at room temperature (RT), unless otherwise indicated. When water is used in preparing solutions, it is ultra-purified prior to use and is denoted by ddH₂O. ddH₂O has a resistivity of ~ 18 MΩ at 25 °C. Its use decreases contamination risks and promotes consistency. All pH values are measured at RT, unless otherwise noted. Follow institutional guidelines for disposal of all materials.

2.1. Solubilization Screening and Expression Characterization

1. 20X galactose solution: 40% (w/v) in ddH₂O. Autoclave ddH₂0 for 15 minutes and cool to RT. Add galactose and sterile filter (0.2 μm) solution before use (see Note 1).

2. 4X yeast extract peptone galactose (YPG) solution: Dissolve 8% (w/v) yeast extract and 16% peptone (w/v) in ddH₂O. Autoclave 15 minutes and cool to RT. Add galactose from 20X stock to a final concentration of 8% (w/v) and mix (see Note 1).

3. Solubilization buffer: 10% (v/v) glycerol, 20 mM TRIS-HCl, pH 8, 300 mM NaCl, 4 mM β-mercaptoethanol (β-Me), 10 mM imidazole. Store at 4 °C.

4. Detergent of choice. Examples: N-dodecyl-β-D-maltoside (DDM), 2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside (OG-NG), and 2,2-didecyl-1,3-bis-β-D-maltopyranoside (LMNG) (see Note 2).

5. 20X amino acid free yeast nitrogen base (YNB) solution: 13.4% (w/v) yeast nitrogen base (no amino acids) in ddH₂0. Sterile filter (0.2 μm) before use. Store at 4 °C.

6. 10X CSM-His media: 7.9 g/L ddH₂0. Sterile filter (0.2 μm) before use. Store at 4 °C (see Note 3).

7. Synthetic complete without histidine (SC-HIS) media: 37.5 ml 10X CSM-His, 10% (w/v) raffinose, 18.8 ml 20X YNB, and 9.4 ml 40% (w/v) glucose

8. Glucose solution: 40% (w/v) in ddH₂O. Autoclave ddH₂0 for 15 minutes and cool to RT. Add glucose and sterile filter (0.2 μm) before use.

9. 10X raffinose solution: 10% (w/v) in ddH₂O. Autoclave ddH₂0 for 15 minutes and cool to RT. Add raffinose and sterile filter (0.2 μm) before use (see Note 1).

10. 100X protease inhibitor solution (e.g., Halt™ protease cocktail, or equivalent).

11. Lysis buffer: 10% (v/v) glycerol, 50 mM TRIS-HCl, pH 7.4, 10 mM EDTA, 200 mM NaCl. Store at 4 °C.

12. Protein SDS-PAGE standards.

13. SDS-PAGE running buffer: 25 mM TRIS-HCl, pH 8.3, 0.192 M, glycine, 0.1% (w/v) SDS.

14. 4X Laemmli sample buffer: 200 mM TRIS-HCl, pH 6.8, 8% (w/v) SDS, 40% (v/v) glycerol, 50 mM EDTA, 0.08% (w/v) bromophenol blue. Mix thoroughly and make 960 μl aliquots. Store at −20 °C. Add 40 μl β-Me to aliquots immediately before use (see Note 4).

15. Polyacrylamide gel electrophoresis chamber and power supply.

16. Polyacrylamide gel casting chamber

17. N,N,N,N′-tetramethyl-ethylenediamine (TEMED).

18. Ammonium persulfate solution: 10% ammonium persulfate (w/v) in ddH₂O. Store at −20 °C.

19. 30% (w/v) acrylamide/bis-acrylamide: 29:1 acrylamide:bis-acrylamide. Store at 4 °C (see Note 5).

20. Stacking gel buffer: 0.5 M TRIS-HCl, pH 6.8.

21. Resolving gel buffer: 1.5 M TRIS-HCl, pH 8.8.

22. Blotting paper: thin and thick variants.

23. 0.2 μm PVDF membrane.

24. Semidry electrophoretic protein transfer cell.

25. 10X TRIS buffered saline (TBS): 1.5 M NaCl, 0.1 M TRIS-HCl, pH 7.4.

26. 1X TBS with tween (TBST): add 0.05% (v/v) Tween-20 to TBS.

27. Western blot transfer buffer: 25 mM TRIS-HCl, pH 8.3, 192 mM glycine, 20% (v/v) methanol.

28. Blocking solution: 5% (w/v) milk in TBST (see Note 6).

29. Wash buffer: 1X TBS.

30. Antibodies conjugated to fluorescent probe or HRP: purchase or make a POI-specific and an expression tag antibody.

31. Chemiluminescent substrate. Only necessary if using HRP-conjugated antibodies.

32. Lab controller and timer.

33. Bead beater with canister and 0.5 mm glass beads or Avestin Emulsiflex® (C3 or C5).

34. Resuspension buffer: 150 mM NaCl, 1 mM PMSF, 25 mM TRIS-HCl, pH 7.4, 1X protease inhibitor.

35. Thermocycler.

36. Electrophoresis chamber and power supply for agarose gels.

37. Water bath.

38. Incubator shakers.

39. Various centrifuges: high speed, low speed, and microcentrifuges.

40. Gel destaining solution: Combine 120 ml glacial acetic acid, 120 ml methanol, and 460 ml ddH₂O.

41. Coomassie stain: Mix 100 ml of glacial acetic acid, 450 ml of methanol, and 400 ml ddH₂O. Add 1 g Coomassie Brilliant Blue R-250, stir extensively. Use filter paper to remove undissolved matter.

42. Coomassie brilliant blue R-250.

2.2. Immobilized Metal Affinity Purification of Membrane Proteins

1. Chromatography system with fraction collector and absorbance detector (e.g., photodiode array). Purge with ddH₂O after each purification. Flow rates of 0.2-1.0 ml/min for immobilized metal affinity chromatography (IMAC) are typical unless ran under gravity.

2. Nickel affinity column or resin.

3. Immunoblotting components: as in section 2.1.

4. SDS-PAGE components: as in section 2.1.

5. Protein SDS-PAGE standards.

6. Imidazole elution buffer: Mix: 10% (v/v) glycerol, 20 mM TRIS-HCl, pH 8.0 at 4°C, 300 mM NaCl, 4 mM β-Me, 1 mM PMSF, 500 mM imidazole, and detergent (see Note 2). Add β-Me, imidazole, and sterile filter (0.2 μm) before use. Store at 4 °C.

7. Buffer exchange or desalting column.

8. Size exclusion buffer: 150 mM NaCl, 10% glycerol (v/v), 20 mM HEPES, pH 8.0 at 4°C, 1 mM PMSF, 2 mM DTT, and detergent (see Note 2). Add DTT sterile filter (0.2 μm) before use. Store at 4 °C.

9. UV-Vis spectrophotometer.

10. Stripping buffer: combine 1 g SDS, 15 g glycine, and 10 mL Tween 20 in 800 mL ddH₂O. Adjust pH to 2.2 and fill to 1 L with ddH₂O.

11. Affinity tag protease. Chapter example: thrombin. Protease is vector-specific.

2.3. Size Exclusion Chromatography (SEC) Purification

1. Chromatography system with fraction collector and absorbance detector (e.g., photodiode array). Flow rates of 0.4-0.5 ml/min for superdex columns are typical.

2. Superdex 200 Increase 10/300 GL size exclusion column.

3. Immunoblotting components: as in section 2.1.

4. SDS-PAGE components: as in section 2.1.

5. Protein SDS-PAGE standards.

6. Size exclusion buffer: as in section 2.2.

7. UV-Vis spectrophotometer.

8. Spin or stirred cell concentrator.

2.4. Ion Exchange Chromatography Purification

1. Chromatography system with fraction collector and absorbance detector (e.g., photodiode array).

2. Ion exchange columns. Positive or negative charged columns. Column charge must complement POI charge at selected pH. Flow rates of 0.5-1.0 ml/min are typical for ion exchange columns.

3. Immunoblotting components: as in section 2.1.

4. SDS-PAGE components: as in section 2.1.

5. Protein SDS-PAGE standards.

6. Low salt buffer: size exclusion buffer (as in section 2.2) modified to decrease salt concentration as much as possible that does not affect protein stability (typically ~10 mM).

7. UV-Vis spectrophotometer.

3. Methods

Protocols in this chapter have been refined to purify a widening number of MPs while decreasing number of adjustments needed for specific POIs. Protocol steps are derived from empirical practice with specific notes expounding caveats and nuances throughout. Downstream techniques that have used these methods include: proteoliposome and lipid bilayer functional studies (1-3), electron microscopy (4), small angle X-ray scattering (5-7), binding assays (8,9), analytical ultracentrifugation (10), nuclear magnetic resonance (11-13), circular dichroism (14,15), and crystallization studies (16-19).

Although this protocol serves as a useful framework for MP solubilization and purification, the process is fundamentally iterative. During MP purification, experimental feedback dictates impromptu adjustments for target proteins that improve results and yield. Improvements include enhancements in detergent selection, solubilization, homogeneity, monodispersity, overall purity, and reductions of protein-free detergent micelles in final samples. This chapter accommodates the iterative nature of MP purification by noting feedback opportunities, alternatives, and experience-derived suggestions. For a comprehensive look at MP isolation consider reading additional works on purification (20-29).

To avoid potential complications that arise from prokaryotic expression systems, this chapter utilizes S. cerevisiae. Benefits of expressing MPs in S. cerevisiae include: ability to produce various post-translational modifications, robust protein folding machinery inclusive of disulfide bond formation, and cost efficiency, with comparable workflows and ease of genetic manipulation to prokaryotic systems. These benefits relative to more complex, or expensive, eukaryotic systems make S. cerevisiae a preferred starting expression system to overproduce eukaryotic MPs.

3.1. Solubilization Screening and Expression Characterization

Purification processes outlined in this chapter emphasize membrane fraction isolation of S. cerevisiae cultures and detergent screening. Although not an absolute requirement for purification strategies, empirical refinements suggest that excluding membrane fraction isolation decreases the protocol’s broad applicability (30,31). This additional step improves downstream chromatographic separation and purification processes by enriching initial protein mixtures for membrane proteins while discarding many impurities before chromatographic separation begins (see Note 7). It also ensures quantitative target protein solubilization in detergents amenable to downstream methods.

Common MP purification practices require detergent-based extraction from cellular membranes, a process known as solubilization. For yeast expression systems, solubilization is best performed on isolated membrane pellets collected from a high speed post-lysate spin (see Note 8). Concentrations needed for maximal solubilization are dependent on detergents’ critical micelle concentrations (CMCs). Once a detergent reaches its CMC, it begins to self-associate into micelles, large detergent structures responsible for protein solubilization (see Note 9) (24,32). Micelles formed during solubilization are complex detergent, protein, and lipid mixtures. Ideal detergents for target MPs are determined empirically but maltosides and glucosides (e.g., DDM, OGNP, LMNG) are used more often than other detergent types, making them a viable starting point if no information is available on target MP solubilization characteristics (see Notes 6, 10) (33-35).

Another strategy that can improve yields is using multiple detergents. For example, a detergent with a long hydrocarbon chain may efficiently solubilize a target MP, and be exchanged for a short-chain detergent that forms a more compact complex and separates better with chromatographic techniques (24,22). Similarly, a mixed detergent micelle with short and long chains may provide the best overall benefit to solubilization and purification while accommodating downstream methods (e.g., crystallization). Lipids can also be added to micelles to increase solubilization and purification efficiency of target proteins (36). Additionally, bicelles consisting of detergent and lipids can be used for MP solubilization (37-39). If complex micelles, bicelles, or alternative solubilization strategies are required for solubilization of a target protein, detailed information can be found elsewhere (22,24,32,40-49). However, most MPs will sufficiently solubilize in a simple micelle using common frontline detergents such as DDM, OGNG, LMNG, or FC-12. A screening protocol to identify simple micelle solubilization effects for target proteins follows:

1. Prepare a preculture growth.

   1. Place 5 ml sterile SC-His media in a sterile tube.
   2. Inoculate with a single colony from a selective plate containing S. cerevisiae transformed with a desired expression vector.
   3. Incubate at 220 rpm and 30 °C for 24 hours.
2. Prepare a culture growth (see Note 11).

   1. Autoclave a 1L baffled flask containing 280 ml ddH₂0.
   2. Add 95 ml of sterile 4X SC-HIS medium, using a freshly autoclaved 100 ml graduated cylinder.
   3. Inoculate with preculture from step 1c.
   4. Incubate at 220 rpm and 30 °C for 16 hours.
3. Prepare to induce protein expression.

   1. Monitor growth via 600 nm optical density until it is between 15-20.
   2. Add 125 ml of YPG solution to induce protein overexpression.
   3. Continue growing for another 16 hours at 220 rpm and 30 °C (see Note 12).
4. Harvest yeast cells.

   1. Centrifuge growth for 10 minutes at 6,000 X g and 4°C to harvest cells.
   2. Resuspend pellet using 1.5 ml of lysis buffer per g of wet cell pellet mass.
   3. Maintain pellet on ice until lysis or storage to prevent protein degradation.

5. Lyse cells.

   Bead beater lysis:

   • If lysis is not immediately convenient, cells can be stored at −20 °C or −80 °C, for short-term or long-term storage, respectively (see Note 13).

   • Pre-chill bead beater canister of appropriate volume and enough 0.5 mm glass beads to fill it halfway.

   • Add 450 μl of PMSF solution to cell suspension and pour into canister.

   • Fill canister with pre-chilled beads.

   • Operate bead beater for 5 cycles, each cycle alternating between 1-minute maximum speed and 1-minute rest periods. Ensure canister is cooled during runs.

   • Collect approximately 25 ml of lysate (see Note 14) and store on ice.

   Lysis using Avestin C3 or C5 Emulsiflex™:

   1. Add 450 μl of PMSF solution to cell suspension.

   2. Perform 3 passes at 28,000 PSI (25k minimum psi to lyse yeast cells).

   3. Keep lysate on ice.

6. Isolate expressed membrane protein.

   1. Centrifuge crude lysate for 1 hour at 7,500 X g and 4 °C.
   2. Estimate lysis efficiency via ratio of lysed to unlysed cell layers (see Notes 15, 16).
   3. Decant supernatant into an ultracentrifuge tube, avoiding cell debris.
   4. Centrifuge supernatant for 1 hour, at 184,000 X g and 4 °C (see Note 17).
7. Prepare membrane proteins for solubilization screening.

   1. If solubilization is not immediately convenient, membrane pellets can be frozen and stored at −80 °C (see Note 13).
   2. Add 10 μl of protease inhibitor cocktail stock to 0.5 ml of resuspension buffer to make a membrane resuspension buffer (see Note 18).
   3. Resuspend pellet in membrane resuspension buffer using a clean tube. Use a small stir bar for resuspension, spin sample on ice until homogenous (times range from 30 minutes to 3 hours, see Note 19).
   4. If protein solubilization detergent has already been determined, solubilize and proceed to purification testing, step 11.
   5. If protein solubilization has not been determined, proceed to solubilization screening.

8. Prepare a detergent master mix for solubilization screening.

   Add the following reagents to a microcentrifuge tube (see Note 18):

   • 3X solubilization buffer: 100 μl
   • Detergent of choice to final concentration of 10X CMC: 60 μl
   • ddH₂O: 120 μl
9. Prepare solubilized protein samples for high-speed centrifugation.

   1. Aliquot 280 μl of solubilization master mix into separate tubes.
   2. Add 20 μl of resuspended membrane pellet to each tube (see Note 20).
   3. Add a small stir bar to each tube and stir at 4 °C for 1 hour (see Note 21).
   4. Aliquot 30 μl into a PCR tube for SDS-PAGE analysis.
   5. Label tube with sample information, indicating that it is a pre-centrifugation sample.
   6. Store sample on ice until step 12.
10. Perform high-speed centrifugation.

    1. Spin remaining 270 μl of resuspended membrane for 20 minutes at 180,000 X g and 4 °C.
    2. Quickly collect supernatant (solubilized protein) in a clean microcentrifuge tube and mix thoroughly.
    3. Aliquot 30 μl of supernatant into a PCR tube for SDS-PAGE analysis.
    4. Label tube with sample information, indicating that it is a post-centrifugation sample.
11. Prepare SDS-PAGE gels.

    1. Mix a 12% (w/v) resolving gel and pour, using the following components (in order):

       • ddH₂O: 6.6 ml.
       • 30% (w/v) acrylamide mix: 8 ml (see Note 4).
       • 1.5 M Tris-HCl, pH 8.8: 5 ml.
       • 10% (w/v) SDS: 0.2 ml.
       • 10% (w/v) ammonium persulfate: 0.2 ml.
       • TEMED: 8 μl.
       • Required gel percentage may vary depending on POI size.
    2. Prevent dehydration by covering with water, without disturbing gel layer.
    3. After resolving gel has polymerized, decant water. Progress can be estimated by checking surplus gel mixture consistency.
    4. Mix a 5% (w/v) stacking gel (top layer) and pour, using the following components (in order):

       • ddH₂O: 3.4 ml.
       • 30% (w/v) acrylamide mix: 0.83 ml.
       • 1.5 M Tris-HCl, pH 8.8: 0.63 ml.
       • 10% (w/v) SDS: 50 μl.
       • 10% (w/v) ammonium persulfate: 50 μl.
       • TEMED: 5 μl.
       • Required gel percentage may vary depending on POI size.
    5. Carefully place comb into gel, avoiding bubble formation.
    6. Once set, gels can be stored at 4 °C, until needed.
12. Run 2 identical SDS-PAGE gels for each screening condition, if multiple conditions were screened (e.g., different concentrations of a detergent or different detergents). The first gel will be used for immunoblotting and the second will be Coomassie stained as an immunoblot reference.

    1. Mix 960 μl of SDS loading dye with 40 μl of β-Me to make a loading buffer.
    2. Add 10 μl of loading buffer to pre- and post-centrifugation samples.
    3. Incubate samples for 5 minutes at 37 °C.
    4. Normalize samples with loading buffer to their original volumes (see Note 22).
    5. Load 10 μl of each sample and 5 μl of a protein standard into separate wells.
    6. Run gels for 1 hour at 140 V, or until dye front indicates completion.
13. Perform an immunoblot with an SDS-PAGE gel.

    1. Cut 2 pieces of thick and think transfer blotting papers and a 0.2 μm PVDF membrane for every gel run, matching paper and membrane sizes to gels.
    2. Briefly soak membrane in pure methanol.
    3. Soak membrane and transfer papers in transfer buffer for 5 minutes at RT.
    4. Prepare to transfer protein to membrane by stacking the following items, in order, on a semi-dry electrophoretic transfer cell:

       • Thick blot paper.
       • Thin blot paper.
       • PVDF Membrane.
       • SDS-PAGE gel.
       • Thin blot paper.
       • Thick blot paper.
    5. Carefully remove any bubbles that form when preparing for protein transfer by rolling them out with a rolling device or serological pipette.
    6. Run protein transfer for 30 minutes under a constant 0.33 mA, per gel.
    7. Transfer membrane to a container with enough blocking solution to prevent dehydration while blocking.
    8. Block for 1 hour on a rocker.
    9. Prepare a working solution for HRP-conjugated (or fluorescent-conjugated) antibody against POI’s affinity tag, per manufacturer suggestions (see Note 23).
    10. Replace blocking buffer with freshly prepared antibody solution and incubate for 1 hour at RT on a rocker.
    11. Wash membrane 3X on a rocker with TBS buffer at RT, 15 minutes per wash.
    12. Image immunoblot (see Note 24).
14. Coomassie stain an identical SDS-PAGE gel.

    1. Place gel in a container and cover with enough stain to prevent dehydration.
    2. Incubate on a rocker at RT until protein bands become clearly visible (see Note 25).
    3. Transfer gel into fresh ddH₂O.
    4. Use this gel as a reference comparison to the corresponding immunoblot.
15. Analyze immunoblots via densitometry software (see Note 26).

    1. Compare pre- and post-centrifugation samples to quantify tested detergent’s effects.
    2. Compare results between detergents to determine which is best (see Note 27).
    3. If results indicate poor solubilization, consider switching detergents, increasing duration of solubilization, and/or decreasing growth temperatures.
16. Troubleshooting: affinity tag was not detected by immunoblotting.

    1. Potential causes:

       • Unintended protein degradation.
       • Affinity tag is removed by cellular machinery.
       • Inadequate expression duration.
       • Poor expression system codon optimization.
       • Affinity tag blocks protein expression.
       • Cloning error.
       • Poor protein expression.
       • Improper post-translational modifications.
    2. Respective solutions to potential causes:

       • Decrease cell growth temperatures to lower proteolytic events during expression. Add or change protease inhibitors.
       • Use POI-specific antibody to detect expression levels.
       • Increase expression incubation time.
       • Codon-optimize gene of interest to match codon preferences in S. cerevisiae.
       • Move affinity tag to opposite terminus or use a different affinity tag.
       • Sequence expression vector, searching for mutations that could cause frameshifts or alter start/stop codons. Reclone, if needed.
       • Clone an expression tag into N-terminus of vector.
       • Use a different expression system (e.g., HEK293S cells).
17. Troubleshooting: POI pellets with unsolubilized materials after solubilization.

    1. Potential cause:

       • Protein is not sufficiently solubilized by selected detergent.
    2. Solutions to potential cause:

       • Increase solubilization duration.
       • Screen a different detergent.

3.2. IMAC MP Purification Using a Chromatography System

IMAC is often the first step in purification protocols as it is a cost-effective and efficient technique to rapidly separate target proteins from background cellular proteins. IMAC purification exploits the high affinity of an amino acid sequence to a particular metal ion. This chapter leverages affinity of a multiple histidine tag to a nickel column. Imidazole disrupts binding interactions, eluting weakly binding contaminants at low imidazole concentrations while histidine tags require higher imidazole concentrations. This chapter utilizes IMAC but reverse IMAC techniques are also useful for purifying membrane proteins following tag cleavage (50). MP purification generally requires multiple IMAC runs with slight adjustments to imidazole concentrations or number of washes before identifying ideal conditions (see Note 28). For labs producing more than one POI, designating specific affinity columns for each POI can prevent sample cross contamination.

1. Prepare an affinity column for protein loading.

   1. Prepare column at 4 °C, storing and using only buffers maintained at 4 °C.
   2. Equilibrate column using 5 column volumes of buffer determined during solubilization screening, supplemented with 10 mM imidazole (binding buffer).
   3. Equilibrate at suggested flow rate for column in use (typically 0.7-1.2 ml/min).
2. Determine whether POI is solubilizing as expected, from detergent screening (above).

   1. Load supernatant generated when solubilized protein is separated from insoluble material (section 3.1 step 10b) on equilibrated Ni-NTA column (see Note 29).
   2. Save flow-through for analysis (see Note 30).
   3. Store flow-through at 4 °C until it is confirmed to not contain POI (via gel).

3. Wash column with binding buffer until 280 nm reading reaches a steady baseline (see Note 31). Save a 20 μl eluate sample for SDS-PAGE evaluation and store at 4 °C.

4. Reduce column flow rate by half and elute bound protein (see Note 32).

   1. Elute bound protein with a linear gradient of elution buffer, collecting eluate in 0.5-1.0 ml fractions (see Note 33).
   2. Save all fractions with 280 nm absorption peaks. Save 20 μl samples of all peaks for SDS-PAGE evaluation and store at 4 °C.

5. Strip and recharge column per manufacturer suggestions (see Note 34).

6. Analyze samples collected during IMAC purification.

   1. Prepare 2 identical SDS-PAGE gels as described in section 3.1.
   2. Stain one gel and immunoblot the other, as described above.
   3. Pool collected fractions containing POI.
7. Remove imidazole from POI sample.

   1. Prepare a desalting column for buffer exchange by equilibrating per manufacturer directions (see Note 35).
   2. Place 3 ml of POI protein sample on buffer exchange column.
   3. Elute with size exclusion buffer, using manufacturer-suggested volumes (e.g., load 3 ml of sample and elute with 4 ml of buffer).
   4. Repeat steps A-C, as necessary, until imidazole removal is complete.
   5. Pool eluted POI samples, if needed. Save a 20 μl sample for SDS-PAGE evaluation and store at 4 °C.
   6. Measure pooled protein concentration via absorption at 280 nm (see Note 36). Use absorption measurements in the Beer-Lambert equation:

      $$\text{Absorption}=(\text{extinction coefficient}{)}^{\ast }(\text{path length}{)}^{\ast }(\text{protein concentration})$$
8. Cleave and remove affinity tag.

   1. Add appropriate amount of protease to imidazole-free POI sample (see Note 37).
   2. Incubate cleavage reaction as recommended for protease in use (see Note 38).
   3. Save a 20 μl sample of completed cleavage reaction for SDS-PAGE evaluation and store at 4 °C.
   4. Prepare a desalting column for buffer exchange by equilibrating it per manufacturer directions (see Note 39).
   5. Separate protease from sample by running 3 ml of cleaved sample through buffer exchange column.
   6. Elute with size exclusion buffer, using manufacturer-suggested elution volumes.
   7. Repeat steps d-f, as necessary, until protease removal is complete.
   8. Save a 20 μl flow-through sample for SDS-PAGE evaluation. Store it at 4 °C.
9. Analyze samples collected during affinity tag removal.

   1. Prepare 2 identical SDS-PAGE gels as described in section 3.1.
   2. Stain one gel and immunoblot the other, as described above.
   3. Perform an immunoblot with affinity tag antibodies and image the blot.
   4. Strip affinity tag antibody from PVDF membrane.

      • Incubate membrane in stripping buffer for 10 minutes on a rocker at RT.
      • Discard and replace stripping buffer.
      • Incubate for another 10 minutes on a rocker at RT.
      • Wash 2X in PBS, 10 minutes per wash.
      • Wash 2X in PBST, 5 minutes per wash.
   5. Perform a second immunoblot with POI-specific antibody (see Note 40).
10. Troubleshooting: POI is not binding to affinity resin.

    1. Potential causes:

       • Affinity tag is inaccessible to resin.
       • Detergent is not compatible with resin.
    2. Respective solutions to potential causes:

       • Add a spacer region to expression vector between affinity tag and cleavage site.
       • Screen alternative detergents that are resin compatible or incubate solubilization supernatant with resin to batch-bind POI. If these solutions do not work, affinity resin or column may need to be replaced.
11. Troubleshooting: POI elution fractions have high contaminant levels.

    1. Potential cause:

       • Proteins from S. cerevisiae are non-specifically binding resin.
    2. Solutions to potential cause:

       • Incrementally increase initial imidazole concentration in solubilization buffer. As a starting point, increase concentration from 10 mM to 20 mM.
       • Incrementally increase imidazole wash concentration until most contaminants are consistently removed.
12. Troubleshooting: Incomplete affinity tag removal (see Note 41).

    1. Potential causes:

       • Affinity tag is inaccessible because of its location relative to detergent micelle.
       • Affinity tag is inaccessible because of its location relative to protein.
    2. Respective solutions to potential causes:

       • Increase protease concentration by 2- to 4-fold.
       • Add a spacer region to expression vector between affinity tag and cleavage site.
13. Troubleshooting: Protein precipitation occurs during cleavage reaction.

    1. Potential cause:

       • Overall stability or solubility of proteins in the reaction solution decreased.
    2. Solutions to potential cause:

       • Remove precipitated proteins by centrifugation (15,000 x g for 15 minutes at 4 °C) and continue with protocol. Precipitate can be identified by Coomassie staining and immunoblotting. If precipitated protein is POI, altering buffer components (detergent, glycerol, sucrose) may help prevent or decrease precipitation.
14. Troubleshooting: POI is only partially bound to column.

    1. Potential causes:

       • POI binds slowly to column.
       • Initial imidazole concentration is too high.
       • Resin binding capacity has been exceeded.
       • Column needs to be stripped and recharged.
    2. Respective solutions to potential causes

       • Increase POI exposure to column by injecting slower or increase amount of passes POI is run over resin.
       • Decrease imidazole binding concentration.
       • Increase resin volume.
       • Strip and recharge column, per manufacturer instructions. If this has no effect, the column may need to be replaced.

3.3. Modifications for IMAC Purification Without a Chromatography System

IMAC purification can be performed without a chromatography system in batch with defined washing and elution steps at fixed imidazole concentrations. Approaches between gravity and chromatography system separations are similar but gravity separations are more labor-intensive and not amenable to gradient elutions. A major gravity separation benefit is rapid washing and elution at a fixed imidazole concentration (e.g., 500 mM imidazole) to push POI into downstream purification methods. In addition, multiple targets can be simultaneously purified using different gravity columns in parallel. In these instances, best practice suggests allocating resin for specific protein targets to prevent cross-contamination.

1. Prepare a nickel affinity resin slurry according to manufacturer suggestions.

   1. Resin binding capacity may vary by manufacturer.
   2. Prepare slurry in an empty gravity flow chromatography column at 4 °C.
   3. Equilibrate resin using 10 column volumes of buffer determined during solubilization screening, supplementing buffer with 10 mM imidazole. Final resin:buffer ratio of 1:1.
2. Add protein to slurry and allow protein to bind.

   1. Add supernatant generated when solubilized protein is separated from insoluble material (section 3.1 step 10b) to equilibrated slurry.
   2. Nutate at 4 °C for 1 hour followed by passing supernatant over column twice to ensure maximal binding efficiency.
   3. Set aside 20 μl for SDS-PAGE evaluation and store at 4 °C.
3. Wash bound protein with imidazole supplemented buffers (see Note 42).

   1. Set column on a ring stand over clean collection vessels for gravity-flow washes.
   2. Wash with 5 column volumes of buffer from step 1c (see Note 43). Set aside 20 μl of eluate for SDS-PAGE evaluation and store at 4 °C.
   3. Increase imidazole concentration of buffer to 25 mM and wash with 5 column volumes. Set aside 20 μl of eluate for SDS-PAGE evaluation and store at 4 °C.
   4. Increase buffer imidazole concentration to 250 mM and elute with 1 column volume of buffer in 0.5-1.0 ml fractions (see Note 44). Set aside 20 μl for SDS-PAGE from each eluted fraction and store at 4 °C.
   5. Analyze fractions containing protein via Coomassie staining and immunoblotting to verify that POI is present in fractions.

4. Regenerate resin per manufacturer protocol (see Note 45).

5. Follow section 3.2 protocol from imidazole removal until completion.

3.4. Size-Exclusion Chromatography

Size-exclusion chromatography (SEC) is an advantageous chromatographic separation technique to perform after IMAC purification and affinity tag cleavage. SEC separates proteins based on hydrodynamic radius, providing a second dimension to purification. SEC is considered a necessary technique for many protein-based labs because SEC not only contributes to purification but also acts as an analytical tool that can evaluate protein stability, homogeneity, and purity (16-19). Oligomeric state analysis compares retention times between POI and SEC standards while homogeneity is assessed by presence of a single Gaussian peak matching the POI’s native oligomeric state (see Notes 46, 47). Additionally, SEC is often used to exchange buffers leading into follow-on methods such as detergent exchange, removal of protein-free detergent micelles, and ion-exchange chromatography.

1. Equilibrate SEC column with size exclusion buffer. Follow manufacturer directions for maximum pressure, buffer compatibility (e.g., pH on silica-based resins), and flow rates.

2. Run a dilute SEC test sample to determine protein elution pattern.

   1. Prepare an SEC sample by centrifuging an affinity-purified sample (~0.25 mg/ml) for 10 minutes at 4 °C and 10,000 X g (see Note 48).
   2. Measure supernatant protein concentration via 280 nm absorption, as described in section 3.2. Load approximately 0.175 mg POI onto Superdex 200 10/300 GL column. Maximum recommended loading volume for this column is 0.5 ml to ensure proper separation.
   3. Run protein sample at 0.33-0.5 ml/min, monitoring 280 nm absorption to determine which fractions contain protein. Lower flow rates are optimal when buffer viscosity (e.g., inclusion of glycerol) is increased.
   4. Collect eluate in 0.5-1.0 ml fractions.
3. Determine which SEC test sample fractions contain POI.

   1. Coomassie stain SDS-PAGE gels loaded with SEC test sample fractions containing protein, as described above (see Note 49).
   2. Note which fractions contain overexpressed protein.
4. Perform SEC on remaining protein sample that was purified via IMAC.

   1. Load maximal sample amount as many times as needed, until all IMAC purified sample has undergone SEC separation.
   2. Fractions containing POI during each run can be combined (see Note 50).
   3. Combined protein fractions can be concentrated to a desired concentration with a stirred cell concentrator, a spin concentrator, or osmotic pressure (see Notes 51-52). Set aside 0.4 mg of protein for protein characterization (step 5).
   4. Perform SDS-PAGE and immunoblot analysis, as described above on concentrated sample.
   5. If analysis suggests high purity protein and if buffer exchange is not required for functional assays or structural experiments, protein is ready to use.
5. Characterize stability and homogeneity of purified POI.

   1. Load a 0.175 mg sample from step 4c to SEC column. Calculate concentration as described above to determine load volume.
   2. Run separation using original SEC conditions.
   3. Stable/homogeneous proteins should produce identical 280 nm absorption chromatograms as under original conditions (see Note 53).
   4. Remaining sample from step 4c should be stored in different conditions to determine storage effects on stability. Store 0.2 mg of protein at 4 °C and 0.1 mg at −80 °C (see Note 54).
   5. After 24 hours, test half of the protein stored at 4 °C, using the same SEC protocol as before. If current and original chromatograms are identical, protein can be considered stable at 4 °C for 24 hours.
   6. After 1 week, test samples stored at 4 °C and − 80 °C, in a similar manner (see Note 55).
   7. Analyze all fractions containing POI from samples that were stored at 4 °C or −80 °C in step 5 with Coomassie staining and immunoblotting to finalize POI stability and homogeneity characterization at different timepoints.

6. Clean column per manufacturer protocol. Clean every 10-20 separation cycles. Without proper maintenance, column performance and resolution will be affected.

7. Troubleshooting: SEC chromatogram does not show clearly resolved peaks.

   1. Potential causes:

      • Sample is contaminated with other proteins.
      • POI has multiple states of oligomerization.
      • POI is interacting with column packing.
   2. Respective solutions to potential causes:

      • Load a dilute sample to separate contaminants from POI. Concentrated samples broaden peaks, making it difficult to interpret chromatograms. Consider using an orthogonal method such as an anionic or cationic affinity (e.g., Hitrap™ S or Q) or phenyl column.
      • Multiple oligomeric states may not require troubleshooting. Collect fractions corresponding to each oligomeric state and pool them, or not, depending on downstream needs and experiments. If disulfide bonds are causing unnatural oligomerization events, increase reducing agent concentrations. Lastly, columns may need cleaning or replacement.
      • Employ an SEC column with a different type of stationary phase.
8. Troubleshooting: POI is not eluting from column.

   1. Potential causes:

      • Detergent complex is binding or interacting with column matrix.
      • Protein aggregation is causing retention in column or frit.
      • Column has not been adequately maintained.
   2. Respective solutions to potential causes:

      • Use an alternative SEC column.
      • (see Note 56).
      • Clean or replace column.
9. Troubleshooting: POI is eluting in void volume.

   1. Potential causes:

      • Protein aggregation negates retention in column.
      • POI instability in SEC conditions.
   2. Respective solutions to potential causes:

      • (see Note 56).
      • Alter pH and salt concentrations in buffer or try adding an osmolyte in 2.5% (w/v) increments (e.g., sucrose or glycerol).
10. Troubleshooting: POI has an elution pattern that is not Gaussian.

    1. Potential cause:

       • POI complex has more than a single folded conformation.
       • Column has not been adequately maintained.
       • POI is interacting with column’s stationary phase.
    2. Solutions to potential cause (see Note 56):

       • Altering pH and salt concentrations in buffer can also be helpful.
       • Clean or replace column.
       • Employ an SEC column with a different type of stationary phase.
11. Troubleshooting: POI is eluting in multiple peaks.

    1. Potential causes:

       • POI has multiple states of oligomerization that are in equilibrium.
       • OI has multiple states of oligomerization that are not in equilibrium.
    2. Respective solutions to potential cause (see Note 56):

       • Alter buffer pH and salt concentrations or try adding an osmolyte in 2.5% (m/v) increments (e.g. sucrose or glycerol).
       • Determine whether oligomeric states are in equilibrium by repeating SEC on eluted fractions that correspond to a single potential oligomeric state. If peak shape and retention time remain unchanged, oligomers are not in equilibrium. If oligomeric states are not in equilibrium, purify oligomeric state(s) of interest and proceed with downstream applications. If oligomers are in equilibrium, any fraction containing POI will create multiple peaks, corresponding to each oligomeric state, when SEC is repeated.
12. Troubleshooting: POI elutes in void volume if not immediately run on column.

    1. Potential cause:

       • POI is not stable in stored buffer conditions.
    2. Solutions to potential cause (see Note 56):

       • Alter pH and salt concentrations in buffer or try adding an osmolyte in 2.5% (m/v) increments (e.g. sucrose or glycerol).

3.5. Ion Exchange Chromatography

Each protein has an isoelectric point (pI) governed by its amino acid composition. The pI is the specific pH at which average overall protein charge is neutral. Ion exchange chromatography (IEC) exploits pI differences to selectively ionize a POI by adjusting buffer pH to 0.5-1.0 units away from the pI, promoting binding events between a charged POI and column (see Note 57). Buffers should be used at high enough concentrations (25-100 mM) to maintain pH during separation. Salt concentrations must be monitored as well because high ionic strengths can disrupt charge-based interactions between protein and column.

IEC is a flexible purification tool that can be used before, after, or in place of SEC (see Note 58). Apart from purification, IEC can also be used for concentrating protein samples and detergent exchange (see Note 59). IEC is not the focus of this chapter and may not be necessary after an optimized IMAC and SEC purification protocol has been developed. However, a starting point for developing an optimized protocol is provided below. Typically, column selection is an empirical process screening high and low pH IEC columns in pre-equilibrated buffers to assess optimal POI binding interactions with a specified resin.

1. Equilibrate column using low-salt buffer. Equilibrate according to manufacturer suggestions.

2. Ensure that protein buffer is also in low-salt buffer. Dialysis or a salt exchange column may be required for buffer matching.

3. Load protein onto column.

4. Wash column with low-salt buffer.

5. Elute POI using a linear salt gradient up to 1 M concentration (see Note 60). Set flow rate based on manufacturer recommendations for specific columns.

6. Collect and analyze 0.5-1.0 ml fractions with a 280 nm absorption signal.

7. Analyze fractions via SDS-PAGE and immunoblots as described above.

8. Perform analytical SEC on fractions expected to contain POI for verification purposes.

4. Notes

1. Overheated galactose or raffinose solutions isomerize, decreasing protein expression.

2. Detergent concentrations are being screened and must be tested empirically. Example concentrations of common detergents that can serve as starting points are: 40 mM OG, 0.5 mM DDM, 12 mM LDAO, 16 mM CHAPS and 4 mM FC-12. For other detergents start at 1.5-2X the CMC for the buffer conditions (24). Presence of osmolytes (e.g. glycerol) is POI-dependent.

3. Warm ddH₂O can be used if powder is not fully dissolved.

4. SDS causes bubbling. Add SDS last and mix gently. When using stored Laemmli stock, warm solution to dissolve SDS precipitate before use.

5. Unpolymerized acrylamide is a neurotoxin. Handle with protective equipment.

6. Milk in blocking solution spoils at RT, which can introduce contaminants that negatively affect immunoblots if not made fresh or properly stored.

7. Enrichment occurs by separating proteins with high membrane affinity from proteins that poorly associate with membranes. Many proteins that poorly associate with membranes have proteolytic activity and can result in POI degradation if they are not separated.

8. In other expression systems (e.g., HEK293S cells), MPs solubilization can occur directly using tissue homogenization in presence of detergent (51).

9. Micelles functionally resemble lipid bilayers, in terms of MP-stabilizing interactions, which can maintain native protein properties while increasing aqueous solubility for purification.

10. Ideal solubilization is not required if POI remains stable for downstream applications. Quantity can be increased by optimizing POI growth and overexpression conditions. Detailed protocols on expression and optimization can be found in our other chapter, entitled “Overproduction of Membrane Associated, and Integrated, Proteins using Saccharomyces cerevisiae.”

11. This step, and corresponding following steps, can be scaled to increase total protein production by increasing quantity of 1 L baffled flasks or using batch fermentation.

12. Volume in each flask is larger than required for screening purposes but these larger volumes are recommended to increase reproducibility when upscaling. Small volume expression protocols are notorious for not scaling in a predictable manner. If the ADE2 mutation is present in S. cerevisiae, they will turn pink before cell harvesting.

13. Stopping here can affect protein stability. Storage tolerance must be empirically determined for each POI. Thaw frozen pellets on ice if lysing cells from storage,

14. 25 ml is a cost-efficient way to screen expression levels. If maximal protein recovery is desired, glass beads and canister can be rinsed with lysis buffer until all cells are collected.

15. Top white layer of pellet is lysed material and bottom brown (or pink if ADE2 cells are used) are unlysed cells. Alternatively, lysate can be analyzed under a microscope.

16. Lysis efficiency should range between 70-95%. Sonication and freeze fracture protocols are inefficient in yeast cells but microfluidizers are an acceptable alternative to bead beaters.

17. This pellet contains proteins that are tightly associated with membranes. Supernatant contains soluble proteins and proteins that only weakly associate with membranes.

18. This is the amount required for a single protein sample. Scale as needed.

19. Expected membrane suspension volume is between 1.5-3.0 ml.

20. Use same ratio of buffer to membrane pellet for every tested detergent. This provides consistent screening results that can be readily compared between detergents or among a single detergent screen of varying concentrations.

21. Ensure that stir bar is active the entire hour. Poor mixing can result in a false negative result.

22. Normalization is critical for accurate comparisons between test conditions and batches.

23. Manufacturers suggest different dilutions, depending on application. Antibodies should be diluted in blocking buffer.

24. HRP-conjugated antibodies should be imaged in a dark room, using an HRP substrate. Antibodies conjugated to fluorescent probes can be imaged directly from membranes with standard immunoblotting hardware or gel imaging hardware with fluorescent channels.

25. If gels are overstained, they can be safely destained using a gel destain solution until stain intensity is lowered to desired amount. Destaining gels can improve signal quality.

26. There is a screening alternative for POIs expressed as fluorescent protein fusions where fluorescent readouts are measured at different stages to analyze initial purification and expression of crude membrane protein. This protocol is available elsewhere (52).

27. Also consider cost:recovery ratio as detergents can represent a large portion of total purification costs.

28. IMAC-purified protein should be carried through remainder of protocol, even if protein yields or purity are initially low. Doing so provides useful information of protein response during downstream purification steps.

29. Sample may be loaded at 0.25 ml/min by a syringe or a pump system and can be passed over a column more than once to increase yields.

30. Monitor protein progression through column via absorption at 280 nm. Set aside 20 μl of flow-through for evaluation and store at 4 °C. Although flow-through should not contain POI, this should be confirmed via staining and immunoblotting before discarding sample.

31. On average, this requires 10-15 column volumes.

32. Reduced flow rate decreases POI elution volume.

33. Shallow gradients (e.g., linear elution over 20 column volumes) separates proteins with similar binding strength more effectively than steep gradients but have drawbacks of increased run time and diluted samples.

34. Perform every 5-7 preparations. This removes residual imidazole and proteins that have non-specifically adsorbed to the column. It also prevents loss of binding capacity.

35. For time-efficiency, start equilibrating buffer exchange column during IMAC POI elution.

36. POI extinction coefficients can be reliably predicted using the ExPASy ProtParam tool. Pathlength is cuvette optical length in cm.

37. For a thrombin cleavage site, 4 units of thrombin are needed for each mg of eluted protein. However, amount of protease needed for cleavage varies between proteases.

38. Required incubation time is likely less, but an overnight incubation normally results in full cleavage. To determine minimal cleavage reaction duration, take 10 μl samples of reaction at different time points and run those samples on an SDS-PAGE gel.

39. Effective separation on a desalting column will not be possible if protease has a similar size as expressed protein. For these situations, reverse IMAC or a second affinity column may be required. Depending on size of proteases and POI, dialysis may also be an option for buffer exchange.

40. This serves as check against cleavage reaction since cleaved tag and POI should not colocalize. Analysis of all collected samples during IMAC will reveal if protein did not process as anticipated during purification. If that occurs, troubleshooting will be required to fully assess.

41. Some detergents may inhibit protease activity and detergent micelles can sterically hinder proteases from accessing cleavage recognition sites (53,54). If protease activity appears to be inhibited, consider adding more protease (up to 10-fold recommended amount). If protease is not inhibited by detergent but is still performing poorly, changing tag location from one terminus to the other may resolve cleavage issues. Adding a flexible linker sequence immediately preceding cleavage sites can also solve this problem by increasing space between them.

42. If contamination is high, a 5 column volume 40 mM imidazole wash can be added before elution. If this step is added, set aside 20 μl of eluate for SDS-PAGE evaluation and store at 4 °C.

43. If IMAC yields are lower than expected, running flow-through of this step over column twice, followed by another wash of 5 column volumes may improve yields.

44. Until a protocol is finalized, this wash will need to be collected in 0.5-1.0 ml fractions that are checked for 280 nm absorption values to determine which fractions contain protein. After a protocol is finalized, POI should consistently elute at the same point. Protocols can then be adjusted to collect just fractions of interest.

45. Between each use, resin should be regenerated to remove residual imidazole and proteins that have non-specifically bound resin.

46. Protein standards may not accurately represent POI molecular weight because POI is purified as a complex, which can affect hydrodynamic radius during SEC separation (32). Expanded hydrodynamic radii cause an exaggerated molecular weight appearance relative to controls. Another consideration is that detergent, protein, or lipid present in micelle complexes can have electrostatic or hydrophobic interactions with SEC matrix, which can also misrepresent MP molecular weights (55). Potential electrostatic interactions between protein complex and matrix are mitigated by performing SEC with salt but addition of salt alone may not be enough to overcome electrostatic interactions. Thus, having a variety of available SEC columns with unique matrices is recommended.

47. Although such conditions are strong supporting evidence of homogeneity, they do not prove that a given sample is fully homogeneous (56-58). Analytical ultracentrifugation is a more powerful technique that can more accurately resolve homogeneity of an isolated protein.

48. Centrifugation is critical for removing protein precipitates and aggregates, if present, which lead to poor column performance, increased back pressure, and potentially expensive repairs.

49. Fractions without an observable 280 nm signal do not need to be tested. Immunoblots can also be run to increase confidence protein identity.

50. POI is assumed to elute at same point as dilute sample run.

51. Osmotic pressure is applied by transferring protein sample to dialysis tubing with a low enough cut off that POI will not be lost. Dialysis tubing is placed on dry PEG, that is too large to enter dialysis tubing, on a clean rocking container until volume reaches a targeted amount. To determine a target volume, concentration of pooled fractions must be known prior to applying osmotic pressure (e.g., if pooled fractions are half the desired concentration, target volume is half of starting volume). Absorption spectroscopy can be used to determine sample concentrations before, and after, applying osmotic pressure.

52. Detergent micelles can concentrate with protein which can affect functional and structural characterization techniques. Dialysis can combat detergent overconcentration and should be performed against a buffer containing minimum detergent amounts to prevent precipitation. In general, detergents dialyze slower than proteins and detergents with low CMCs dialyze slower than detergents with high CMCs. Dialysis is not strictly required but dialysis ensures that detergent concentrations are consistent between batches and during characterization, which is very important for assays that are detergent sensitive. An alternative method is to rebind purified protein to an ion exchange column, flush with buffer, and elute concentrated protein in a smaller number of fractions. This approach will significantly reduce concentration fold of protein-free detergent micelles.

53. Peak height will likely change but time of elution and general shape should remain constant.

54. If freezing samples with liquid nitrogen, its impact on stability should be tested.

55. Stability can be assessed in this manner for any time frame. For general lab convenience, it is encouraged to test two additional 4 °C storage samples at 2 and 3 weeks of storage. A sample stored at −80 °C for one or two months could provide high laboratory flexibility, if protein stability is retained.

56. Screen a detergent within the same class but with a longer hydrocarbon tail. If extending the tail does not work, screen a new class of detergents

57. pI estimates can be obtained from the ExPASy ProtParam tool.

58. If SEC is not run as a purification technique it should still be performed for analytical purposes, as discussed in section 3.5.

59. IEC allows for tight protein binding and small elution volumes to concentrate samples. This should be accompanied by analytical SEC to determine its effects on stability. Detergent exchange is performed by binding POI to the column and heavily washing in a buffer with an alternative detergent at a pH and salt concentration that does not disrupt binding.

60. With optimization, a step gradient can be used to remove contaminants with binding affinities lower than POI’s affinity before POI is eluted in a linear fashion.