Journal: International Journal of Molecular Sciences

Article Title: The Mitochondrial Small Heat Shock Protein HSP22 from Pea is a Thermosoluble Chaperone Prone to Co-Precipitate with Unfolding Client Proteins

doi: 10.3390/ijms21010097

Figure Lengend Snippet: Binding of the hydrophobic probe bis-8-anilinonaphthalene-l-sulphonic acid (bis-ANS) to HSP22rec, rhodanese, and fumarase. Proteins were used at a final concentration of 0.5 µM, calculated on the monomer molecular mass basis for multimeric proteins. They were incubated for 15 min at the indicated temperature (23 or 55 °C). The fluorescent probe bis-ANS was added immediately after heating at a saturating concentration of 37 µM. Each condition was tested at least in triplicate. Fluorescence values are indicated in relative fluorescence unit (RFU). Fluorescence measurements for HSP22 were not significantly different after incubation at 55 °C compared to 23 °C (t-test α= 0.05; p -value = 0.071).

Article Snippet: Bovine liver rhodanese (essentially salt free, purchased from Merck KGaA, Darmstadt, Germany) was diluted in 20 μL of 50 mM MOPS pH 7.5, 1 mM EDTA, and 1 mM DTT to a final concentration of 75 ng/μL alone or with nine-fold molar excess of either recombinant HSP22 or lysozyme (Merck KGaA, Darmstadt, Germany).

Techniques: Binding Assay, Concentration Assay, Incubation, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: The Mitochondrial Small Heat Shock Protein HSP22 from Pea is a Thermosoluble Chaperone Prone to Co-Precipitate with Unfolding Client Proteins

doi: 10.3390/ijms21010097

Figure Lengend Snippet: Thermal aggregation of rhodanese at 50 °C in the presence of thermostable proteins. Rhodanese (Rho) (10 µg) was heated 20 min at 50 °C alone or in the presence of bovine serum albumin (BSA) or HSP22rec (HSP) at a 1:1 mass ratio. After centrifugation, equal fraction volumes of supernatants and pellets were analyzed by SDS-PAGE, and proteins were revealed by colloidal blue staining.

Article Snippet: Bovine liver rhodanese (essentially salt free, purchased from Merck KGaA, Darmstadt, Germany) was diluted in 20 μL of 50 mM MOPS pH 7.5, 1 mM EDTA, and 1 mM DTT to a final concentration of 75 ng/μL alone or with nine-fold molar excess of either recombinant HSP22 or lysozyme (Merck KGaA, Darmstadt, Germany).

Techniques: Centrifugation, SDS Page, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Mitochondrial Small Heat Shock Protein HSP22 from Pea is a Thermosoluble Chaperone Prone to Co-Precipitate with Unfolding Client Proteins

doi: 10.3390/ijms21010097

Figure Lengend Snippet: Thermal aggregation of rhodanese in the presence of HSP22rec ( a ) or lysozyme ( b ). HSP22 rec (HSP22) or lysozyme (Lys) were mixed with rhodanese (Rho) using different molar ratio. Samples were heated for 20 min at 50 °C and then centrifuged. Equal fraction volumes of soluble and pellet fractions were analyzed by SDS-PAGE, and proteins were revealed by colloidal blue staining.

Article Snippet: Bovine liver rhodanese (essentially salt free, purchased from Merck KGaA, Darmstadt, Germany) was diluted in 20 μL of 50 mM MOPS pH 7.5, 1 mM EDTA, and 1 mM DTT to a final concentration of 75 ng/μL alone or with nine-fold molar excess of either recombinant HSP22 or lysozyme (Merck KGaA, Darmstadt, Germany).

Techniques: SDS Page, Staining