Beta amylase

Manufactured by Merck Group

Sourced in United States

Beta-amylase is an enzyme that catalyzes the hydrolysis of starch, glycogen, and related polysaccharides by cleaving off successive maltose units from the non-reducing ends of the substrate. It is a key component in various industrial and laboratory applications.

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10 protocols using beta amylase

Mass Photometry Characterization of Nanobodies

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Select nanobodies were binned using mass photometry (MP). Experiments were performed on a Refeyn OneMP instrument (Refeyn Ltd). The instrument was calibrated with a mix of BSA (Sigma-Aldrich), thyroglobulin (Sigma-Aldrich) and beta-amylase (Sigma-Aldrich). Coverslips (Thorlabs) and gaskets (Grace Bio-Labs) were prepared by washing with 100% IPA followed by ddH₂O, repeated 3 times, followed by drying with HEPA filtered air. 12 μl of buffer was added to each well to focus the instrument after which 8 μl of protein solution was added and pipetted up and down to briefly mix after which movies/frame acquisition was promptly started. The final concentration in each experiment of recombinant Spike S1 monomer (Sinobiological) and each nanobody was 30 nM and between 25–40 nM respectively. Movies were acquired for 60 s (6000 frames) using AcquireMP (version 2.3.0; Refeyn Ltd) using standard settings. All movies were processed, analyzed, and masses estimated by fitting a Gaussian distribution to the data using DiscoverMP (version 2.3.0; Refeyn Ltd).

Mast F.D., Fridy P.C., Ketaren N.E., Wang J., Jacobs E.Y., Olivier J.P., Sanyal T., Molloy K.R., Schmidt F., Rutkowska M., Weisblum Y., Rich L.M., Vanderwall E.R., Dambrauskas N., Vigdorovich V., Keegan S., Jiler J.B., Stein M.E., Olinares P.D., Hatziioannou T., Sather D.N., Debley J.S., Fenyö D., Sali A., Bieniasz P.D., Aitchison J.D., Chait B.T, & Rout M.P. (2021). Nanobody Repertoires for Exposing Vulnerabilities of SARS-CoV-2. bioRxiv.

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Characterization of Biomolecular Interactions

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Cytochrome c (12.4 kDa)
from equine heart, myoglobin (17.6 kDa) from equine heart, pepsin
(35 kDa) from porcine gastric mucosa, bovine serum albumin (BSA, 66.4
kDa), conalbumin (77 kDa) from chicken egg white, concanavalin A (102
kDa) from Canavalia ensiformis, immunoglobulin
G (IgG, ∼150 kDa) from human serum, beta amylase (β-amylase,
223.8 kDa) from sweet potato, chaperonin 60 (GroEL, ∼800 kDa)
from Escherichia coli, ammonium acetate,
tris-acetate, potassium chloride, ethylenediaminetetraacetic acid
(EDTA), adenosine-5′-triphosphate (ATP), magnesium chloride,
ammonium hydroxide, and acetic acid were all purchased from Sigma-Aldrich
(Zwijndrecht, The Netherlands). Methanol, acetone, and LC-MS grade
water were purchased from Biosolve (Valkenswaard, The Netherlands).
Nanospray needles were homemade from preheated borosilicate glass
capillaries (Science Products GmbH, Hofheim, Germany) on a DMZ universal
electrode puller (Zeitz-Instruments Vertriebs GmbH, Munich, Germany)
followed by gold coating with a SC7640 sputter coater (Quorum Technologies,
Kent, UK).

Mathew A., Buijs R., Eijkel G.B., Giskes F., Dyachenko A., van der Horst J., Byelov D., Spaanderman D.J., Heck A.J., Porta Siegel T., Ellis S.R, & Heeren R.M. (2021). Ion Imaging of Native Protein Complexes Using Orthogonal Time-of-Flight Mass Spectrometry and a Timepix Detector. Journal of the American Society for Mass Spectrometry, 32(2), 569-580.

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Cellulose Hydrolysis Enzyme Assay

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The reagents p-nitrophenyl β-d-glucopyranoside (ρNPβGlu), methylumbelliferyl-β-glucopyranoside (MUβGlu), octyl-β-glucopyranoside (octylβGlu), gentiobiose, laminaribiose, cellobiose, cellotriose, cellotetraose, cellopentaose, microcrystalline cellulose (Avicel^®, Cat 11365), low viscosity carboxymethylcellulose (CMC, Cat C5678), ρ-nitrophenyl-β-d-galactopyranoside (ρNPβgal), methylumbeliferil-β-d-mannopyranoside (MUβMan), ρ-nitrophenol, 4-methylumbelliferone, cellulases from T. reesei ATCC 26921 (Cat. C8546) and the molecular markers Blue dextran (Cat. D4772), Cytochrome C (Cat. 12 kDa), carbonic anhydrase (Cat. C7025), serum albumin bovine (Cat. A8531), alcohol dehydrogenase (Cat. A8656), and beta amylase (Cat. A8781) were purchased from Sigma-Aldrich Company (St. Louis, Missouri, USA). Trypsin (Cat. V511A) for mass spectrometry was acquired from Promega Corporation (Madison, Wisconsin, USA). Glucose oxidase-based glucose quantitation reagent was acquired from Bioclin (Minas Gerais, Brazil). Other reagents used in this work were analytical grade.

da Costa S.G., Pereira O.L., Teixeira-Ferreira A., Valente R.H., de Rezende S.T., Guimarães V.M, & Genta F.A. (2018). Penicillium citrinum UFV1 β-glucosidases: purification, characterization, and application for biomass saccharification. Biotechnology for Biofuels, 11, 226.

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Purification of Proteins by Size Exclusion

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Size exclusion chromatography was done manually. A size exclusion column was packed using 45 ml of Sephacryl S-200 HR (Sigma-Aldrich) beads. The void volume (24 ml) of this column was determined by running blue dextran. 1mg of protein (in 100 μl volume) was loaded on the column equilibrated with the buffer containing 20mM HEPES-KOH pH7.6, 100mM KCl and 10% Glycerol. Fractions of 50 μl were collected and the absorbance at 280 nM were recorded. Protein fractions were also analyzed on 12% SDS-PAGE. Gel filtration standard consisted of a mixture of Beta-amylase (200 kDa), Alcohol Dehydrogenase (150 kDa), Bovine Serum Albumin (66 kDa) and Carbonic Anhydrase (29 kDa) bought from Sigma.

Poornima G., Mythili R., Nag P., Parbin S., Verma P.K., Hussain T, & Rajyaguru P.I. (2019). RGG-motif self-association regulates eIF4G-binding translation repressor protein Scd6. RNA Biology, 16(9), 1215-1227.

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Site-specific Labeling of A11 cMb with Maleimide-Cy5.5

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A11 cMb was site-specifically labeled with maleimide-Cy5.5 (mal-Cy5.5) by selective reduction of and conjugation to C-terminal cysteines. In a typical reaction, 200 µg of protein at 1 mg/mL in phosphate-buffered saline (PBS) was reduced using a 2-fold molar excess of tris(2-carboxyethyl)phosphine (TCEP, Pierce) for 30 min at room temperature. Equimolar mal-Cy5.5 (Amersham, GE Healthcare) was added to the reduced A11 cMb for 2 h at room temperature. Excess mal-Cy5.5 was removed using a Micro Bio-Spin™ Size Exclusion Columns (Bio-Rad) pre-equilibrated with PBS. Dye-to-protein ratio (D:P) was determined by measuring the protein (280 nm) and Cy5.5 absorbance (675 nm) with a spectrophotometer (NanoDrop 2000). Successful conjugation was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC).
For SEC, a Superdex 200 10/30 GL column (GE Healthcare) was used with an ÄKTA purifier (GE Healthcare) with PBS as the mobile phase (0.5 mL/min). Absorbance at 280 nm (protein) and 675 nm (Cy5.5) was recorded. The following protein standards were used: beta-amylase (200 kDa), bovine serum albumin (66 kDa), and carbonic anhydrase (29 kDa) (Sigma).

Tsai W.K., Zettlitz K.A., Tavaré R., Kobayashi N., Reiter R.E, & Wu A.M. (2018). Dual-Modality ImmunoPET/Fluorescence Imaging of Prostate Cancer with an Anti-PSCA Cys-Minibody. Theranostics, 8(21), 5903-5914.

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Polystyrene Beads, Amylase, and HSP90 Protocol

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We studied the following analytes for the experiments: 20nm polystyrene beads (Thermo-Fisher)- 0.05% w/v; beta-amylase (Sigma) −0.05% w/v; Heat Shock Protein 90 (HSP90) (Obtained from the Hugel Lab)- 50 nMol. All analytes were diluted in 1X PBS that was additionally buffered with 5nM of MgCL in the case of the HSP90.

Yang W., van Dijk M., Primavera C, & Dekker C. (2021). FIB-milled plasmonic nanoapertures allow for long trapping times of individual proteins. iScience, 24(11), 103237.

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Determine Oligomeric State of BifR Protein

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To determine oligomeric state of BifR, a Superdex 75 5/150 GL column with mobile phase buffer consisting of 50 mM Tris (pH 7.0) and 150 mM NaCl was used, and the column was calibrated with markers carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), beta amylase (200 kDa), and blue dextran (2000 kDa) (Sigma). A standard curve was obtained as a plot of V_e/V_o as a function of the log₁₀ of molecular weight (where V_e and V_o represent the retention volume of the protein and void volume of the column, respectively).^{20 (link)} Reduced protein (with DTT) or protein oxidized with CuCl₂ was run on the column.

Gupta A., Fuentes S.M, & Grove A. (2017). Redox-Sensitive MarR Homologue BifR from Burkholderia thailandensis Regulates Biofilm Formation. Biochemistry, 56(17), 2315-2327.

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Purification of Ovine Haptoglobin Fractions

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Ag5 fraction was obtained by Fast Protein Liquid Chromatography (FPLC) procedures, performed on an AKTA Explorer 10 system (GE Healthcare). Aliquots of 5 mL of 5 sheep HCF were desalted by HiPrep 26/10 column (GE Healthcare), lyophilized, solubilized in 500 µL of 50 mM phosphate buffer at pH 7.6, containing 150 mM NaCl (running buffer) and filtered at 12,000×g on PVDF 0.22 µm filters (Ultrafree-MC Centrifugal Filter Units, Millipore). Size exclusion chromatography fractionations were then performed on a Superdex-200 column (10/300 GL, GE Healthcare). Runs were performed at room temperature at a flow rate of 1 mL/min. Column was calibrated, under the same conditions, with standard proteins (thyroglobulin, apoferritin, beta-amylase, BSA and carbonic anhydrase, Sigma Aldrich).

Pagnozzi D., Biosa G., Addis M.F., Mastrandrea S., Masala G, & Uzzau S. (2014). An Easy and Efficient Method for Native and Immunoreactive Echinococcus granulosus Antigen 5 Enrichment from Hydatid Cyst Fluid. PLoS ONE, 9(8), e104962.

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SEC Analysis of EPS Samples

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SEC was performed using (i) a liquid chromatograph with pump (LC-20AD), (ii) a fluorescence detector (RF-20AXS), (iii) an auto sampler (SIL-20AHT), and (iv) a communication module (CMB-20A). The molecular weights (MW) of the EPS samples were estimated by passing 15 μL of the filtered samples through an analytical scale SEC column (OHpak SB-804 HQ) following its guard column (OHpak SB-G). Tris buffer (25 mM Tris, pH 7.0 ± 0.1) was used as the mobile phase. The SEC column was calibrated using transferrin, serum albumin bovine, myoglobin and beta amylase obtained from Sigma Aldrich (the details are provided in Supporting Information (SI)).
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Wong L.L., Natarajan G., Boleij M., Thi S.S., Winnerdy F.R., Mugunthan S., Lu Y., Lee J.M., Lin Y., van Loosdrecht M., Law Y., Kjelleberg S, & Seviour T. (2020). Extracellular protein isolation from the matrix of anammox biofilm using ionic liquid extraction. Applied microbiology and biotechnology, 104(8).

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Nanobody Mass Profiling by MP

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Select nanobodies were binned using MP. Experiments were performed on a Refeyn OneMP instrument (Refeyn Ltd). The instrument was calibrated with a mix of BSA (Sigma-Aldrich), thyroglobulin (Sigma-Aldrich), and beta-amylase (Sigma-Aldrich). Coverslips (Thorlabs) and gaskets (Grace Bio-Labs) were prepared by washing with 100% IPA followed by _ddH₂O, repeated three times, followed by drying with HEPA filtered air. 12 μl of buffer was added to each well to focus the instrument after which 8 μl of protein solution was added and pipetted up and down to briefly mix after which movies/frame acquisition was promptly started. The final concentration in each experiment of recombinant spike S1 monomer (Sino Biological) and each nanobody was 30 nM and between 25 and 40 nM, respectively. Movies were acquired for 60 s (6000 frames) using AcquireMP (version 2.3.0; Refeyn Ltd) using standard settings. All movies were processed, analyzed, and masses estimated by fitting a Gaussian distribution to the data using DiscoverMP (version 2.3.0; Refeyn Ltd).

Mast F.D., Fridy P.C., Ketaren N.E., Wang J., Jacobs E.Y., Olivier J.P., Sanyal T., Molloy K.R., Schmidt F., Rutkowska M., Weisblum Y., Rich L.M., Vanderwall E.R., Dambrauskas N., Vigdorovich V., Keegan S., Jiler J.B., Stein M.E., Olinares P.D., Herlands L., Hatziioannou T., Sather D.N., Debley J.S., Fenyö D., Sali A., Bieniasz P.D., Aitchison J.D., Chait B.T, & Rout M.P. (2021). Highly synergistic combinations of nanobodies that target SARS-CoV-2 and are resistant to escape. eLife, 10, e73027.

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