Hannah Arbach, Caley Butler, and Kathryn A. Mcmenimen*

Supplementary Information

Journal: Cell Stress and Chaperones

Chaperone Activity of Human Small Heat Shock Protein-GST Fusion Proteins

Hannah Arbach, Caley Butler, and Kathryn A. McMenimen*

Department of Chemistry, Mount Holyoke College, 50 College Street, South Hadley, MA 01075

*To whom correspondance should be addressed: Dr. Kathryn A. McMenimen, Department of Chemistry, Mount Holyoke College, South Hadley, MA 01075, Telephone (413-538-3375), Email:

Table of Contents

Figure CaptionPage

SI.Fig. 1. Western blot of GST-HspB1.3

SI. Fig. 2. Size exclusion chromatography protein standards.3

SI. Fig. 3. Size exclusion chromatography of 12 M GST-sHsps.4

SI. Fig. 4. Western blot analysis of GST-HspB5.4

SI. Fig. 5. Chaperone assay control data.5

SI. Table 1 Change in intrinsic tryptophan fluorescence with temperature.5

SI. Fig. 1 Western blot of GST-HspB1.Native PAGE electrophresis was used prior to western blotting. A monoclonal Anti-GST antibody was used in the top image and only GST-HspB1 and GST were observed. Both monomers and dimers of GST-HspB1 were identified. A monoclonal antibody was used in the bottom image for HspB1. Monomers and dimers of GST-HspB1 were observed. For all samples, 5 M of each protein was loaded.

SI. Fig 2 Size exclusion chromatography protein standards. Size exclusion chromatography of molecular weight standards eluted (0.3 mL/min.) from a Superdex 200 10/300 GL column in PBS pH 7.4 for calibration. The peaks are assigned as follows: 1. aggregate peaks (>670kD), 2. thyroglobulin (669 kDa), 3. -globulin(158 kDa), 4. ovalbumin (44 kDa), 5. myoglobin (17 kDa), 6. vitamin B12(1.35 kDa).

SI. Fig. 3 Size exclusion chromatography of GST-sHsps. GST-HspB1 (black) and GST-HspB5 (blue) eluted from a Superdex 200 10/300 GL column in PBS at pH 7.4. Concentrations of both GST-sHsps are 12 M. The column and samples are equilibrated at 4°C.

SI. Fig. 4Western blot analysis of GST-HspB5. SDS-PAGE analysis on a 4-20% gel was performed prior to blotting with Anti-HspB5 and Anti-GST antibodies. Both the purified (lane 1) and crude (lane 2) protein samples were identified.

SI. Fig. 5 Chaperone assay control data. Light scattering at 340 nm was measured to indicate the aggregation propensity of each sample at 45°C for 1 hour. MDH only (orange), CS only (red), GST + CS (purple) and GST + MDH (green) exhibited significant aggregation. All other samples, GST (green), GST-HspB1 (pink), and GST-HspB5 (blue) did not aggregate or scatter light upon extended heating at 45°C.

SI. Table 1Change in intrinsic tryptophan fluorescence with temperature. Tryptophans are present in GST, HspB1, and HspB5, as well as the substrate proteins, CS and MDH.

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