Performing a Purity and Homogeneity Check

Purity check

As with water-soluble proteins, SDS-PAGE is the most widespread method for assessing the purity of membrane proteins. Coomassie Blue, silver staining, or Deep PurpleTM (for fluorescence) can be used for detection. The LaemmLi system is commonly used. Some modifications may be necessary for membrane proteins, as outlined below.

Boiling of the sample with SDS can cause aggregation of membrane proteins. As an alternative to boiling, incubation at 60 °C for 30 min or at 37 °C for 60 min are useful starting points for preparing the sample for SDS-PAGE. On the other hand, some membrane proteins are fully compatible with boiling and boiling may be required for complete solubilization with SDS.

Membrane proteins frequently do not move according to the expected molecular weight in SDS-PAGE. They often move faster (i.e., appear smaller) possibly due to incomplete unfolding or due to binding more SDS per mass unit protein as compared with a water-soluble protein.

SDS-PAGE Clean-Up kit has successfully been used to treat samples containing interfering detergent or that are too dilute for SDS-PAGE. With this kit, proteins are quantitatively precipitated while interfering substances remain in solution.

Size homogeneity characterization

Protein aggregation is a common issue with membrane proteins. Aggregation often appears to be irreversible and it may occur slowly over time but also rapidly and unexpectedly with modest changes in ionic strength, pH, protein:detergent ratio, and other factors. For membrane proteins, it is as important to keep track of aggregation as it is to monitor protein activity.

Aggregation may not always be detected by SDS-PAGE since SDS solubilizes most aggregates. Gel filtration is the method of choice for rapid detection of aggregation and it can be applied under a wide variety of conditions. It is widely used as an efficient assay to assess the size homogeneity in purified membrane protein samples.

Gel filtration allows detection of relatively small changes in the size of detergent-protein complexes when different detergents are compared. Gel filtration is often used to give an indication of the suitability of different detergents for a particular protein. Separation with gel filtration is thus an important tool for qualifying the membrane protein preparation for further analysis. As an example, gel filtration is very often used for assessing the suitability of different detergents for membrane protein crystallization. In some cases, however, membrane proteins have been found to crystallize under conditions where the protein preparation did not look homogenous.

Rapid size homogeneity characterization of membrane proteins by gel filtration with Superdex 200 5/150 GL

Material

Column: Superdex 200 5/150 GL (CV = 3 mL)
Buffer: Buffers in the pH range 3 to 12 can be used. An ionic strength above 15 mS/cm is recommended. Include the selected detergent(s) at a concentration above the CMC.

Procedure

1. Equilibrate the column with 2 CV of buffer.
2. Allow the protein to equilibrate in the buffer to avoid aggregation or disaggregation of the detergent-membrane protein complex during the gel filtration run. Check that the sample is not turbid. If it is, centrifuge at 10 000 × g for 10 min.
3. Apply the sample (4 to 50 µL) and perform the separation at a flow rate of 0.6 mL/min. Back pressure must not exceed 1.5 MPa. The run will take approximately 6 min. (Fig 1.7)

Figure 1.7. Screening of pH and ion strength conditions for optimal homogeneity and stability of a detergent-protein complex. Rapid gel filtration with Superdex 200 5/150 GL showed a symmetrical peak when the separation was performed at pH 5.2 in 0.1 M NaCl (A), indicating a homogenous protein under these conditions. At somewhat higher salt concentration (D) a small peak appeared close to the void volume, indicating that oligomerization or aggregation appeared to a limited extent. At both pH 7.5 and pH 9.5 significant peaks were obtained close to the void volume, indicating severe oligomerization or aggregation. The complete screening procedure was achieved in only a few hours, including the time for column equilibration. Sample consumption was 6 × 10 µL for the complete screen. Data kindly provided by Dr. Said Eshagi, Karolinska Institute, Stockholm, Sweden.

Size homogeneity characterization of membrane proteins by gel filtration with Superdex 200 10/300 GL

Material

Column: Superdex 200 10/300 GL (CV = 24 mL)
Buffer: Buffers in the pH range 3 to 12 can be used. An ionic strength above 15 mS/cm is recommended. Include the selected detergent(s) at a concentration above the CMC.

Procedure

1. Equilibrate the column with 2 CV of buffer.
2. Allow the protein to equilibrate in the buffer to avoid aggregation or disaggregation of the detergent-membrane protein complex during the gel filtration run. Check that the sample is not turbid. If it is, centrifuge at 10 000 × g for 10 min.
3. Apply the sample (25 to 250 µL) and perform the separation at a flow rate of 0.75 mL/min. Back pressure must not exceed 1.5 MPa. The run will take approximately 40 min.

It is possible to achieve the same information at a slightly lower resolution in only 6 min using a Superdex 200 5/150 GL column (Figure 1.7).

Size homogeneity analysis of purified membrane proteins using Superdex 200 10/300 GL is shown in Figure 1.13.

Figure 1.13. Size homogeneity characterization of membrane proteins by gel filtration. IMAC purifications of three E. coli membrane proteins were analyzed by gel filtration using Superdex 200. Each protein was purified separately by IMAC in the presence of two different detergents. Gel filtration analysis was done with the same detergent as used for purification. A symmetric peak (which is not at the void volume), as in chromatogram C, shows that the protein did not aggregate, indicating that purification was successful. Blue line = A280 trace. Used with permission. Copyright 2005 The Protein Society.

Charge homogeneity characterization

High-resolution anion exchange chromatography is widely used for purification of membrane proteins, often as a second chromatography step after IMAC. It is also an excellent technique for charge homogeneity characterization of purified membrane proteins. Figure 1.14 shows charge homogeneity characterization of fractions obtained from the IMAC purification of histidine-tagged cytochrome bo3 ubiquinol oxidase, a membrane protein complex, overexpressed in E. coli. The first IMAC fraction gave a shouldered peak on Mono Q (left). Heterogeneity was confirmed by SDS-PAGE. The second IMAC fraction gave a single sharp, symmetrical peak on Mono Q. Homogeneity was confirmed by SDS-PAGE which showed four bands corresponding to the expected subunit molecular weights of 58 000, 33 000, 22 000, and 17 000.

Figure 1.14. Charge homogeneity characterization of histidine-tagged cytochrome bo3 ubiquinol oxidase using anion exchange chromatography with Mono Q. Two fractions obtained from IMAC purification were analyzed. Electrophoretic analysis was done with PhastSystemTM using PhastGelTM 8-25% and silver staining. M = Low Molecular Weight Calibration Kit; E = detergent extract of E. coli membranes; FT = flowthrough material; F1 = fraction 1 from HiTrap Chelating HP 1 mL; F2 = fraction 2 from HiTrap Chelating HP 1 mL. See application note 18-1128-92 ”Purification and chromatographic characterization of an integral membrane protein.”