Catalase

Manufactured by GE Healthcare

Sourced in Spain, United States

Catalase is a common enzyme found in nearly all living organisms exposed to oxygen, where it functions to catalyze the decomposition of hydrogen peroxide to water and oxygen. It is a very important enzyme in protecting cells from oxidative damage by reactive oxygen species.

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Lab products found in correlation

8 protocols using catalase

Molecular Mass Determination of F. plautii DAEase

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The subunit molecular mass of F. plautiiDAEase was determined by SDS-PAGE under denaturing conditions, using a ladder of pre-stained proteins (MBI fermentas, Hanover, MD, USA) as references. All protein bands were stained with Coomassie Blue for visualization. The molecular mass of the native enzyme was investigated by gel-filtration chromatography using a Sephacryl S-300 preparative-grade column HR 16/60 (GE Healthcare, Piscataway, NJ, USA). The purified enzyme was loaded onto the column and eluted with 50 mM Tris-HCl (pH 7.5) buffer containing 150 mM NaCl at a flow rate of 1mL/min. The column was calibrated with ferritin (440 kDa), catalase (206 kDa), aldolase (158 kDa), and conalbumin (75 kDa) as reference proteins (GE Healthcare). The molecular mass of the native enzyme was calculated by comparing with the migration length with that of the reference proteins.

Park C.S., Kim T., Hong S.H., Shin K.C., Kim K.R, & Oh D.K. (2016). D-Allulose Production from D-Fructose by Permeabilized Recombinant Cells of Corynebacterium glutamicum Cells Expressing D-Allulose 3-Epimerase Flavonifractor plautii. PLoS ONE, 11(7), e0160044.

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Invaplex Purification and Characterization

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A TSK-Gel G5000PW_XL 7.8-mm by 30-cm column (Tosoh Bioscience) with a 10-µm particle size and an exclusion limit of 1 × 10⁷ Da was calibrated using blue dextran (2 MDa), thyroglobulin (669 kDa), catalase (232 kDa), ovalbumin (43 kDa), and RNase A (13.7 kDa) (all from GE Healthcare) in 0.02 M Tris-HCl, 0.5 M NaCl, pH 9.0 (Invaplex_AR buffer), connected to a Shimadzu 10ADvp HPLC system. S. flexneri 2a Invaplex_AR (17.5 µg) and Invaplex_NAT (70 µg) were applied in separate runs under the same conditions at a flow rate of 0.5 ml/min and 400-lb/in² maximum pressure. Chromatographic traces were recorded both at 280 nm using an SPD-10Avp UV detector and at 215 nm using the SPD-M10Avp photodiode array.

Turbyfill K.R., Clarkson K.A., Vortherms A.R., Oaks E.V, & Kaminski R.W. (2018). Assembly, Biochemical Characterization, Immunogenicity, Adjuvanticity, and Efficacy of Shigella Artificial Invaplex. mSphere, 3(2), e00583-17.

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Protein Purification and Characterization

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Protein (1 mg/ml; 400 ul) was applied to a Superdex 75 analytical column (30 × 1 cm; GE-Healthcare) using AKTA Explorer (GE-Healthcare) and eluted at a flow rate of 0.8 ml/min in buffer PBS monitoring absorbance at 280, 260 and 220 nm. Molecular weight standards catalase (232 kDa), aldolase (163 kDa), BSA (67 kDa), OvoAlbumin (44 kDa), Chymotrypsinogen A (25 kDa) and RNaseA (13.7 kDa) (GE-Healthcare) were used. For CD40-FasL containing medium the molecular weight standards are as presented in Additional file 2: Figure S2A.

Aronin A., Amsili S., Prigozhina T.B., Tzdaka K., Shen R., Grinmann L., Szafer F., Edebrink P., Rauvola M.A., Shani N, & Elhalel M.D. (2014). Highly efficient, In-vivo Fas-mediated Apoptosis of B-cell Lymphoma by Hexameric CTLA4-FasL. Journal of Hematology & Oncology, 7, 64.

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Size Exclusion Chromatography of C-LrtA

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SEC experiments were performed at pH 8.0 (50 mM Tris) and 0.250 M NaCl in a Superose 12 10/300 GL column, which was connected to an AKTA FPLC (GE Healthcare, Barcelona, Spain); absorbance at 280 nm was monitored during the elution. Flow rate was 0.8 mL/min, and C-LrtA was loaded in a volume of 100 μL at several protein concentrations, in the range of 30–500 μM (in protomer concentration). The markers used to calibrate the column were ferritin, catalase, aldolase, albumin, bovine RNase A and blue dextran (GE Healthcare, Barcelona, Spain); the isolated protein markers were loaded in the column in the above described buffer, at the same flow rate, and three independent measurements were performed with each marker.

Neira J.L., Giudici A.M., Hornos F., Arbe A, & Rizzuti B. (2018). The C Terminus of the Ribosomal-Associated Protein LrtA Is an Intrinsically Disordered Oligomer. International Journal of Molecular Sciences, 19(12), 3902.

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Fractionation of HEK293T cell extracts

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HEK293T cells were incubated with Roeder A buffer (22 (link)) in an appropriate amount for 10 min at room temperature, dounced 10 times, and adjusted to 150 mM NaCl. After centrifugation at 17 000 g for 30 min the supernatants (S100 extracts) were filtrated with Millex-HA, 0.45 μm filter unit (Merck Millipore) and applied to a Superdex 200 HiLoad 16/600 or Superdex 200 increase 10/300 GL column (GE Healthcare). 2 ml respectively 0.5 ml fractions were collected in running buffer (150 mM NaCl, 50 mM Tris/HCl pH 7.5) and analyzed by immunoblotting. The columns were calibrated with thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa), and RNase (14 kDa) (GE Healthcare).

Schmitz K., Cox J., Esser L.M., Voss M., Sander K., Löffler A., Hillebrand F., Erkelenz S., Schaal H., Kähne T., Klinker S., Zhang T., Nagel-Steger L., Willbold D., Seggewiß S., Schlütermann D., Stork B., Grimmler M., Wesselborg S, & Peter C. (2021). An essential role of the autophagy activating kinase ULK1 in snRNP biogenesis. Nucleic Acids Research, 49(11), 6437-6455.

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Blue Native PAGE for Protein Complexes

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Blue Native (BN)-PAGE was performed based on the method of Schagger and colleagues^{30 (link)} with minor modifications. Briefly, mitochondria (100 μg protein) were solubilized for 15 min with digitonin using a 6g/g digitonin/protein ratio. Insoluble material was removed by centrifugation at 21,000 g for 30 min at 4 °C, the soluble component was combined with BN–PAGE loading dye and separated on a 3–13% acrylamide–bisacrylamide precast BN-PAGE gel. For separation, cathode buffer (15 mM bis-Tris [pH 7.0] and 50 mM tricine) containing 0.02% (w/v) Coomassie Blue G was used until the dye front had reached approximately one-third of the way through the gel before exchange with cathode buffer lacking Coomassie Blue G. Anode buffer contained 50 mM bis-Tris (pH 7.0). Native complexes were separated at 4 °C at 110V for 1 hour, followed by 12mA constant current. Thyroglobulin (669 kDa), ferritin (440 kDa), Catalase (232 kDa), Lactate dehydrogenase (140 kDa), and bovine serum albumin (BSA 67 kDa) were used as markers (GE Healthcare).

Momcilovic M., Jones A., Bailey S.T., Waldmann C.M., Li R., Lee J.T., Abdelhady G., Gomez A., Holloway T., Schmid E., Stout D., Fishbein M.C., Stiles L., Dabir D.V., Dubinett S.M., Christofk H., Shirihai O., Koehler C.M., Sadeghi S, & Shackelford D.B. (2019). In vivo imaging of mitochondrial membrane potential in non-small cell lung cancer. Nature, 575(7782), 380-384.

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Gel Filtration Chromatography Assay

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The gel filtration chromatography assays were performed using a DuoFlowTM chromatography system with a HiLoad 10/60 Superdex 200 column (GE Healthcare, WI, USA). The column was equilibrated with 50 mM Tris-HCl buffer (pH 7.4) containing 300 mM NaCl, and 0.5 mM EDTA. Protein samples (5 mg) were loaded onto the column for fractionation. Blue dextran (2000 kDa), thyroglobulin (699 kDa), ferritin (440 kDa), catalase (232 kDa), and ovalbumin (43 kDa) (GE Healthcare, WI, USA) were used as the standard proteins.

Lee S., Jia B., Liu J., Pham B.P., Kwak J.M., Xuan Y.H, & Cheong G.W. (2015). A 1-Cys Peroxiredoxin from a Thermophilic Archaeon Moonlights as a Molecular Chaperone to Protect Protein and DNA against Stress-Induced Damage. PLoS ONE, 10(5), e0125325.

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Purification of Protein Complexes from MCF7 Cells

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MCF7 cells were lysed in ice cold gel filtration lysis buffer [50 mM HEPES, pH 7.4; 150 mM NaCl; 0.5% NP-40; 1X EDTA-free Complete Protease Inhibitor Cocktail (Roche)] at 4°C with gentle rotation for 30 min. Lysates were then centrifuged twice at 15,000 rpm for 15 min to remove debris. 2.5 mg of lysate was then fractionated by size-exclusion chromatography at 4°C, using a Superdex 200 10/300 GL column, an AKTA Purifier system and Unicorn 5.1 software (GE Healthcare). Protein complexes were eluted using column buffer [25 mM HEPES, pH 7.4; 150 mM NaCl], and 24 fractions of 1 mL each were collected. Equal volumes of each of the different fractions were then analyzed by western blotting. The column was calibrated with the following molecular weight standards: Blue Dextran 2000, thyroglobulin, ferritin, catalase, aldolase, bovine serum albumin, ovalbumin, and ribonuclease A (GE Healthcare). Based on the Blue Dextran 2000 elution profile, the void volume was determined to be 9 mL, in agreement with the manufacturer's predicted void volume.

Swetzig W.M., Wang J, & Das G.M. (2016). Estrogen receptor alpha (ERα/ESR1) mediates the p53-independent overexpression of MDM4/MDMX and MDM2 in human breast cancer. Oncotarget, 7(13), 16049-16069.

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