Journal: PLoS ONE

Article Title: Crystal structure of a cold-active protease (Pro21717) from the psychrophilic bacterium, Pseudoalteromonas arctica PAMC 21717, at 1.4 Å resolution: Structural adaptations to cold and functional analysis of a laundry detergent enzyme

doi: 10.1371/journal.pone.0191740

Figure Lengend Snippet: Substrate-binding mode of Pro21717-CD. (A) Stereoview of the substrate-binding site in Pro21717-CD. The bound, co-purified peptide (magenta) facilitated the characterization of the substrate-binding pocket and the substrate-binding mode of Pro21717. The side chains of the catalytic triad (Asp185, His244, and Ser425) and the residues in the nearby S2 and S4 pockets are presented as a stick model. (B) Ribbon diagram showing superposition of Pro21717-CD (orange) and Carlsberg subtilisin (cyan) at the active site. The A–A–P–F peptide in Pro21717-CD and the A–C–T–L peptide in subtilisin Carlsberg are shown as sticks. The catalytic triad residues Asp185, His244, and Ser425 (Pro21717-CD) and Asp32, His64 and Ser221 (subtilisin Carlsberg) are also shown as sticks. (C) Schematic view of the A–A–P–F peptide-binding mode in Pro21717-CD. The key hydrogen and hydrophobic interactions involved in peptide binding are presented using Ligplot.

Article Snippet: In comparison with a commercial detergent enzyme, Pro21717-CD showed resistance and stability that was similar to subtilisin Carlsberg (the activity of which was also slightly reduced by LAS and SDS), although Cu2+ and H2 O2 enhanced the activity of subtilisin Carlsberg but not Pro21717-CD.

Techniques: Binding Assay, Purification

Journal: PLoS ONE

Article Title: Crystal structure of a cold-active protease (Pro21717) from the psychrophilic bacterium, Pseudoalteromonas arctica PAMC 21717, at 1.4 Å resolution: Structural adaptations to cold and functional analysis of a laundry detergent enzyme

doi: 10.1371/journal.pone.0191740

Figure Lengend Snippet: Structural comparison of Pro21717-CD with other serine proteases. (A) Superimposition of Pro21717-CD (orange color) onto AprB2 protease (an extracellular protease from Dichelobacter nodosus ; PDB code: 3LPC; gray color) revealed that Pro21717-CD adopts a novel conformation in the capping-loop region of the active site. (B) Structural comparison of Pro21717-CD (orange) and subtilisin Carlsberg (green) complexed with the protease inhibitor OMTKY3 (cyan). Notably, the conformation of the co-purified peptide in the active site of Pro21717-CD is identical to that of the residues (Ala15[P4], Cys16[P3], and Thr17[P2]) in the subtilisin Carlsberg inhibitor (OMTKY3) (right panel).

Article Snippet: In comparison with a commercial detergent enzyme, Pro21717-CD showed resistance and stability that was similar to subtilisin Carlsberg (the activity of which was also slightly reduced by LAS and SDS), although Cu2+ and H2 O2 enhanced the activity of subtilisin Carlsberg but not Pro21717-CD.

Techniques: Protease Inhibitor, Purification

Journal: PLoS ONE

Article Title: Crystal structure of a cold-active protease (Pro21717) from the psychrophilic bacterium, Pseudoalteromonas arctica PAMC 21717, at 1.4 Å resolution: Structural adaptations to cold and functional analysis of a laundry detergent enzyme

doi: 10.1371/journal.pone.0191740

Figure Lengend Snippet: Activity and stability assays for Pro21717-CD. (A–D) Thermal stability and the effect of pH on Pro21717-CD activity (black circles), compared with those for subtilisin Carlsberg (open circles). The optimal temperature (A) and pH (C) for activity were measured, and the thermal stability of Pro21717-CD and subtilisin Carlsberg (B), and the activity in various pH values (D) were examined as described in the Materials and Methods section. Briefly, the thermal melting profile of Pro21717-CD and subtilisin Carlsberg were recorded using CD spectroscopy. The signal at 220 nm was recorded while the temperature was increased from 5 to 95°C at intervals of 2.5°C. (E) The effects of metal ions and detergents on Pro21717-CD activity and stability. (F) Effect of recombinant protease Pro21717-CD on milk/blood/ink stain removal. The washing test was performed at 15°C in LAS detergent in water (control) or LAS detergent containing 13 mg of Pro21717-CD in water (reaction).

Article Snippet: In comparison with a commercial detergent enzyme, Pro21717-CD showed resistance and stability that was similar to subtilisin Carlsberg (the activity of which was also slightly reduced by LAS and SDS), although Cu2+ and H2 O2 enhanced the activity of subtilisin Carlsberg but not Pro21717-CD.

Techniques: Activity Assay, Spectroscopy, Recombinant, Staining

Journal: Applied and Environmental Microbiology

Article Title: Cold-Active Serine Alkaline Protease from the Psychrotrophic Bacterium Shewanella Strain Ac10: Gene Cloning and Enzyme Purification and Characterization

doi:

Figure Lengend Snippet: Effect of temperature on the activities of rSapSh (•) and subtilisin Carlsberg (■) toward azocasein.

Article Snippet: The activity at pH 11.0 was more than 80% of the activity at pH 9.0, a finding similar to the findings obtained with subtilisins Carlsberg and BPN′ ( ).

Techniques:

Journal: Applied and Environmental Microbiology

Article Title: Cold-Active Serine Alkaline Protease from the Psychrotrophic Bacterium Shewanella Strain Ac10: Gene Cloning and Enzyme Purification and Characterization

doi:

Figure Lengend Snippet: (A) Thermal stabilities of rSapSh (•) and subtilisin Carlsberg (■). The enzymes were incubated at 60°C in 50 mM Tris-HCl buffer (pH 9.0) supplemented with 2 mM CaCl 2 for different periods of time, and the residual activities were determined at 60°C with AAPF as the substrate. (B) Denaturation of rSapSh (•) and subtilisin Carlsberg (■) by urea. The enzymes were incubated with various concentrations of urea for 30 min, and then the reactions were started by adding AAPF.

Article Snippet: The activity at pH 11.0 was more than 80% of the activity at pH 9.0, a finding similar to the findings obtained with subtilisins Carlsberg and BPN′ ( ).

Techniques: Incubation

Journal: Journal of molecular catalysis. B, Enzymatic

Article Title: Effect of prolonged exposure to organic solvents on the active site environment of subtilisin Carlsberg

doi: 10.1016/j.molcatb.2010.01.021

Figure Lengend Snippet: Fluorescence emission spectra of dansyl PEG-subtilisin Carlsberg in ( A ) 1,4-dioxane, and ( B ) in acetonitrile. Day 0; day 1; day 2; day 3; day 4. (excitation at 360 nm).

Article Snippet: Effect of PEG modification on subtilisin Carlsberg activity, enantioselectivity, and structural dynamics in 1,4-dioxane.

Techniques: Fluorescence

Journal: Journal of molecular catalysis. B, Enzymatic

Article Title: Effect of prolonged exposure to organic solvents on the active site environment of subtilisin Carlsberg

doi: 10.1016/j.molcatb.2010.01.021

Figure Lengend Snippet: Emission spectra of Dansyl-labeled subtilisin Carlsberg during incubation in 1,4-dioxane under controlled water activity. Day 0; day 1; day 2; day 3; day 4. Excitation at 360 nm.

Article Snippet: Effect of PEG modification on subtilisin Carlsberg activity, enantioselectivity, and structural dynamics in 1,4-dioxane.

Techniques: Labeling, Incubation, Activity Assay

Journal: Molecules

Article Title: Further Stabilization of Alcalase Immobilized on Glyoxyl Supports: Amination Plus Modification with Glutaraldehyde

doi: 10.3390/molecules23123188

Figure Lengend Snippet: ( A ) Hydrolysis of casein by different preparations of Alcalase at 69 °C and pH 9. Reactions were performed using 0.005 mg of enzyme/mL. Other specifications are described in Methods section. Open circles, dashed line: Free enzyme; Solid triangles: Immobilized Alcalase; Solid Squares: Immobilized, aminated and glutaraldehyde modified enzyme; ( B ) Operational stabilities of different Alcalase preparations at 69 °C and pH 9. Other reaction conditions as in Figure 8 A. Solid triangles: Immobilized Alcalase; Triangles, solid line: Immobilized, aminated and glutaraldehyde modified enzyme.

Article Snippet: Alcalase, a protease solution supplied by Novozyme, has subtilisin Carlsberg as main component and it is produced by a strain of Bacillus licheniformis .

Techniques: Modification

Journal: Molecules

Article Title: Further Stabilization of Alcalase Immobilized on Glyoxyl Supports: Amination Plus Modification with Glutaraldehyde

doi: 10.3390/molecules23123188

Figure Lengend Snippet: Operational stability of immobilized, aminated and glutaraldehyde treated Alcalase in hydrolysis of casein at pH 9. The experiments were performed at 45 (triangles) or 50 °C (squares). Other experimental conditions are described in methods.

Article Snippet: Alcalase, a protease solution supplied by Novozyme, has subtilisin Carlsberg as main component and it is produced by a strain of Bacillus licheniformis .

Techniques:

Journal: Molecules

Article Title: Further Stabilization of Alcalase Immobilized on Glyoxyl Supports: Amination Plus Modification with Glutaraldehyde

doi: 10.3390/molecules23123188

Figure Lengend Snippet: 3D surface structure models of Alcalase (PDB code 1SBC). The 3D structures were obtained from the Protein Data bank (PDB) and displayed using Pymol software version 0.99rc6.

Article Snippet: Alcalase, a protease solution supplied by Novozyme, has subtilisin Carlsberg as main component and it is produced by a strain of Bacillus licheniformis .

Techniques: Software

Journal: Molecules

Article Title: Further Stabilization of Alcalase Immobilized on Glyoxyl Supports: Amination Plus Modification with Glutaraldehyde

doi: 10.3390/molecules23123188

Figure Lengend Snippet: Courses of hydrolysis of casein at pH 9 and 50 (circles) or 60 °C (triangles) catalysed by free (empty symbols, dashed line) or immobilized Alcalase (solid symbols, solid line). The concentration of enzyme (free or immobilized) was 0.005 mg/mL in all cases. Other conditions may be found in methods section.

Article Snippet: Alcalase, a protease solution supplied by Novozyme, has subtilisin Carlsberg as main component and it is produced by a strain of Bacillus licheniformis .

Techniques: Concentration Assay

Journal: Molecules

Article Title: Further Stabilization of Alcalase Immobilized on Glyoxyl Supports: Amination Plus Modification with Glutaraldehyde

doi: 10.3390/molecules23123188

Figure Lengend Snippet: ( A ) Immobilization course of Alcalase in 4% glyoxyl agarose beads. Immobilization was performed at pH 10 and 25 °C as described in methods. Open circles: reference; Solid triangles: supernatant; Solid squares; suspension. ( B ) Inactivation courses of free (open circles) and immobilized (closed triangles) Alcalase. Inactivation was performed at 54 °C and pH 7. Other details are stated in methods section.

Article Snippet: Alcalase, a protease solution supplied by Novozyme, has subtilisin Carlsberg as main component and it is produced by a strain of Bacillus licheniformis .

Techniques:

Journal: Molecules

Article Title: Further Stabilization of Alcalase Immobilized on Glyoxyl Supports: Amination Plus Modification with Glutaraldehyde

doi: 10.3390/molecules23123188

Figure Lengend Snippet: Effect of glutaraldehyde modification on the stability of immobilized Alcalase. The enzyme was modified at pH 7 with 0.1% ( v / v ) for 1 h, washed and incubated 3 h at pH 8 before reduction. Inactivation was performed at pH 7 and 54 °C. Other specifications are described in methods. Glx-AL: open circles; Glx-AL-GLU: solid triangles.

Article Snippet: Alcalase, a protease solution supplied by Novozyme, has subtilisin Carlsberg as main component and it is produced by a strain of Bacillus licheniformis .

Techniques: Modification, Incubation

Journal: Food Technology and Biotechnology

Article Title: A Systematic Approach to the Comparison of Cost Efficiency of Endopeptidases for the Hydrolysis of Atlantic Salmon (Salmo salar) By-Products

doi: 10.17113/ftb.54.04.16.4553

Figure Lengend Snippet: Effect of proteolytic enzyme activity on the reduction of pH at t =50 °C during hydrolysis by Alcalase 2.4L at enzyme activity in units (U) per g of substrate of 5.5–88 U/g

Article Snippet: The endopeptidases used were Alcalase 2.4L and Neutrase 0.8L (Novozymes, Bagsvćrd, Denmark), Corolase 7089 (AB Enzymes, Darmstadt, Germany), Promod 671L (Biocatalysts, Cardiff, UK) and Protex 7L (DuPont, Wilmington, DE, USA).

Techniques: Activity Assay

Journal: Food Technology and Biotechnology

Article Title: A Systematic Approach to the Comparison of Cost Efficiency of Endopeptidases for the Hydrolysis of Atlantic Salmon (Salmo salar) By-Products

doi: 10.17113/ftb.54.04.16.4553

Figure Lengend Snippet: Response surface plots for: a) degree of hydrolysis (DH) of Alcalase 2.4L, and b) protein recovery (PR) based on regression models given in

Article Snippet: The endopeptidases used were Alcalase 2.4L and Neutrase 0.8L (Novozymes, Bagsvćrd, Denmark), Corolase 7089 (AB Enzymes, Darmstadt, Germany), Promod 671L (Biocatalysts, Cardiff, UK) and Protex 7L (DuPont, Wilmington, DE, USA).

Techniques:

Journal: BioMed Research International

Article Title: Identification of a New Serine Alkaline Peptidase from the Moderately Halophilic Virgibacillus natechei sp. nov., Strain FarDT and its Application as Bioadditive for Peptide Synthesis and Laundry Detergent Formulations

doi: 10.1155/2019/6470897

Figure Lengend Snippet: (a) Effect of organic solvents on the stability of the purified rSAPV, SPVP, and Thermolysin type X. Various organic solvents, with different log  P values (50%, v/v), were tested at 190 strokes per min and 37°C for 3 days to evaluate their effects on peptidase stability. The residual peptidase activities were assayed under the same conditions of each enzyme. The activity of the enzyme without any organic solvent was taken as 100%. The activity is expressed as a percentage of activity level in the absence of organic solvents. Each point represents the mean of three independent experiments. Performance evaluation of the purified SAPV peptidase. (b) Stability of rSAPV, Alcalase 2.4 L FG, and Bioprotease N100L in the presence of liquid and solid laundry detergents. The list of liquid detergents included: Class (EJM, Sfax, Tunisia), EcoVax and Dipex (Klin Productions, Sfax, Tunisia), Skip (Unilever, France), and Nadhif (Henkel-Alki, Tunisia). The solid detergents used were OMO (Unilever, France), Det (Sodet, Sfax, Tunisia), Dixan (Henkel-Alki, Tunisia), iSiS (Henkel, Algiers, Algeria), and Ariel (Procter Gamble, Switzerland). Peptidase activity of the control sample, which contained no additive and incubated under similar conditions, was taken as 100%. Vertical bars indicate standard error of the mean ( n  = 3). Washing performance analysis of rSAPV and commercial enzymes (500 U/mL) using egg (c), blood (d), and chocolate (e) stains with Class detergent (7 g/L). Stained cloths were washed with tap water (A), Class detergent (B), Class added with Alcalase 2.4 L FG (C), Class added with Bioprotease N100L (D), and Class added with rSAPV (E). I: untreated clothes (control) and II: treated clothes.

Article Snippet: With casein as substrate, the k cat /K m of rSAPV was at least 2.6, 10.3, 7.9, and 9.8 folds higher, respectively, than those observed for SPVP, Thermolysin type X, Alcalase 2.4 L FG, and Bioprotease N100L, while with Suc-F-A-A-F-p NA as the substrate, the k cat /K m of rSAPV was at least 1.9, 9.3, 6.9, and 9.2 folds higher, respectively, than those observed for SPVP, Thermolysin type X, Alcalase 2.4 L FG, and Bioprotease N100L ( ).

Techniques: Purification, Activity Assay, Incubation, Staining

Journal: BMC Biotechnology

Article Title: On the activity loss of hydrolases in organic solvents: II. a mechanistic study of subtilisin Carlsberg

doi: 10.1186/1472-6750-6-51

Figure Lengend Snippet: Effect of water (0.5 % v/v) and controlled water activity during incubation of 4-EFPO-TEMPO-labeled Subtilisin Carlsberg in 1,4-dioxane by EPR Spectroscopy.

Article Snippet: We have previously reported that the activity of the serine protease subtilisin Carlsberg is significantly reduced upon storage in several organic solvents, irrespective of its preparation, hydration, hydrophobicity of the solvent, the reaction temperature and of the substrates used [ ].

Techniques: Activity Assay, Incubation, Labeling, Electron Paramagnetic Resonance, Spectroscopy

Journal: BMC Biotechnology

Article Title: On the activity loss of hydrolases in organic solvents: II. a mechanistic study of subtilisin Carlsberg

doi: 10.1186/1472-6750-6-51

Figure Lengend Snippet: Storage stability of lyophilized subtilisin Carlsberg in 1,4-dioxane: (○) neat, (●) with 0.5% H 2 O added, and (▲) controlling water activity with BaBr 2 salts.

Article Snippet: We have previously reported that the activity of the serine protease subtilisin Carlsberg is significantly reduced upon storage in several organic solvents, irrespective of its preparation, hydration, hydrophobicity of the solvent, the reaction temperature and of the substrates used [ ].

Techniques: Activity Assay

Journal: BMC Biotechnology

Article Title: On the activity loss of hydrolases in organic solvents: II. a mechanistic study of subtilisin Carlsberg

doi: 10.1186/1472-6750-6-51

Figure Lengend Snippet: Storage stability of subtilisin Carlsberg co-lyophilized with methyl-β-cyclodextrin in 1,4-dioxane: (○) neat, (●) with 0.5% H 2 O added, and (▲) controlling water activity with BaBr 2 salts.

Article Snippet: We have previously reported that the activity of the serine protease subtilisin Carlsberg is significantly reduced upon storage in several organic solvents, irrespective of its preparation, hydration, hydrophobicity of the solvent, the reaction temperature and of the substrates used [ ].

Techniques: Activity Assay

Journal: Nutrients

Article Title: Endotoxin-Binding Peptides Derived from Casein Glycomacropeptide Inhibit Lipopolysaccharide-Stimulated Inflammatory Responses via Blockade of NF-κB activation in macrophages

doi: 10.3390/nu7053119

Figure Lengend Snippet: Inhibitory activity of casein glycomacropeptide (GMP) and its hydrolysates on NO production in LPS-stimulated RAW264.7 macrophages. ( A ) Effect of GMP hydrolysates obtained with alcalase, pepsin and papain at various hydrolysis periods on NO production in LPS-stimulated RAW264.7 macrophages; ( B ) Effect of different GMP hydrolysates on NO production in LPS-stimulated RAW264.7 macrophages. GMP hydrolysates produced by alcalase at 6 h hydrolysis, pepsin at 1 h hydrolysis and papain at 1 h hydrolysis were referred to as GHA, GHE and GHP, respectively. ( C ) LPS-stimulated NO production in RAW264.7 macrophages was suppressed by GHP in a dose-dependent manner. The results are presented as the means ± SD of four independent experiments. Means with different letters are significantly different from each other at p

Article Snippet: Alcalase (EC 3.4.21.62, with an activity of 2.4 Anson units per gram; endoproteinase from Bacillus licheniformis ) was provided by Novo Nordisk Biochem.

Techniques: Activity Assay, Produced

Journal: Journal of structural biology

Article Title: Understanding specificity of the mycosin proteases in ESX/type VII secretion by structural and functional analysis

doi: 10.1016/j.jsb.2013.09.022

Figure Lengend Snippet: Structure based sequence alignment of MycP 1mth , MycP 3msm and subtilisin Carlsberg

Article Snippet: Structural comparison of two serine proteinase-protein inhibitor complexes: eglin-c-subtilisin Carlsberg and CI-2-subtilisin Novo.

Techniques: Sequencing

Journal: Journal of structural biology

Article Title: Understanding specificity of the mycosin proteases in ESX/type VII secretion by structural and functional analysis

doi: 10.1016/j.jsb.2013.09.022

Figure Lengend Snippet: Catalytic triad and binding grooves of MycP 1mth , MycP 3msm and subtilisin Carlsberg/eglin complex

Article Snippet: Structural comparison of two serine proteinase-protein inhibitor complexes: eglin-c-subtilisin Carlsberg and CI-2-subtilisin Novo.

Techniques: Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Water dynamics and salt-activation of enzymes in organic media: Mechanistic implications revealed by NMR spectroscopy

doi: 10.1073/pnas.0601113103

Figure Lengend Snippet: Dependence of ln( k cat / K M ) app on the correlation times of mobile deuterons, (τ c ) D 2 O , for salt-activated subtilisin Carlsberg in hexane (○) and acetone (•). Thelinearity of each plot reflects an Arrhenius-type dependence assuming

Article Snippet: For this reason, we have investigated the dynamics of enzyme-associated water using a well-studied model enzyme, the bacterial protease subtilisin Carlsberg (SC). shows that the total water content of salt-activated SC decreased linearly with the percent NaF content.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Water dynamics and salt-activation of enzymes in organic media: Mechanistic implications revealed by NMR spectroscopy

doi: 10.1073/pnas.0601113103

Figure Lengend Snippet: Catalytic efficiency, ( k cat / K M ) app (•), of salt-activated subtilisin Carlsberg in hexane; % water (wt/wt)/% enzyme (wt/wt) (■); integrated NMR peak area normalized for number of scans and amount of sample in arbitrary units (▴).

Article Snippet: For this reason, we have investigated the dynamics of enzyme-associated water using a well-studied model enzyme, the bacterial protease subtilisin Carlsberg (SC). shows that the total water content of salt-activated SC decreased linearly with the percent NaF content.

Techniques: Nuclear Magnetic Resonance

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Water dynamics and salt-activation of enzymes in organic media: Mechanistic implications revealed by NMR spectroscopy

doi: 10.1073/pnas.0601113103

Figure Lengend Snippet: Catalytic efficiency, ( k cat / K M ) app (•), of salt-activated subtilisin Carlsberg in hexane, THF, and acetone in comparison with T 2 (■) of mobile deuterons as a function of dielectric constant of solvent.

Article Snippet: For this reason, we have investigated the dynamics of enzyme-associated water using a well-studied model enzyme, the bacterial protease subtilisin Carlsberg (SC). shows that the total water content of salt-activated SC decreased linearly with the percent NaF content.

Techniques:

Journal: Microorganisms

Article Title: Development of Versatile Vectors for Heterologous Expression in Bacillus

doi: 10.3390/microorganisms6020051

Figure Lengend Snippet: Four different subtilisin proteins expressed from FX-compatible Bacillus vectors. ( A ) A representative SDS-PAGE showing the supernatant fraction of the B. subtilis WB800N host after recombinant expression of native subtilisins from B. licheniformis DSM13 (B13), B. paralicheniformis ATCC 9945A (B9945), B. subtilis subsp. subtilis str. 168 (BSU) and B. amyloliquefaciens (BAM). Asterisks indicate recombinant enzymes (matured subtilisins expected mass is 28 kDa). Background represents expression of the empty vector; ( B ) Supernatants in A were screened for proteolytic activity against BODIPY-conjugated casein. Error bars show deviation in three biological replicates; ( C ) Temperature activity profiles of recombinant subtilisins (B13, green; B9945, magenta; BSU, red; BAM, blue) screened for proteolytic activity against the chromogenic succinyl-AAPA peptide, and compared to commercial Alcalase™ 2.4L (orange). Error bars show deviation in three biological replicates; ( D ) pH activity profiles of recombinant subtilisins screened against the peptide in C. Error bars show deviation in three biological replicates.

Article Snippet: Routinely, Alcalase™ 2.4L (Sigma-Aldrich) was used at a dilution 1:10,000 in 8.5 N lysis buffer.

Techniques: SDS Page, Recombinant, Expressing, Plasmid Preparation, Activity Assay

Journal: BMC Cancer

Article Title: Heparan sulfate mediates trastuzumab effect in breast cancer cells

doi: 10.1186/1471-2407-13-444

Figure Lengend Snippet: Glycosaminoglycans profile of SKBR3, MCF7 and MCF7-HPSE1 cells. Sixty percent confluent cells were incubated for 18 hours in serum-free medium containing 150 mCi/ml [ 35 S]-sulphate. Protein-free GAG chains were prepared from the cells and culture medium by incubation with maxatase. Aliquots from the medium and cells were submitted to agarose gel electrophoresis (0.05 M diaminopropane acetate buffer, pH 9.0) and the sulphated GAG identified and quantified as described in methods. (A) , Heparan sulfate (HS) and dermatan sulfate (DS) from SKBR3; (B) , HS and chondroitin sulfate (CS) from MCF7; (C) , HS and DS from MCF7-HPSE1. *P

Article Snippet: Protein free GAG chains were prepared from the cellular fraction (cells plus ECM) and culture medium by incubation with maxatase (Biocon Industrial, Rio de Janeiro, RJ, Brazil; 4 mg/ml) overnight, at 60°C.

Techniques: Incubation, Agarose Gel Electrophoresis