Journal: Biological Procedures Online

Article Title: Dealing with different methods for Kluyveromyces lactis β-galactosidase purification

doi: 10.1251/bpo4

Figure Lengend Snippet: Profiles of β-Galactosidase activity and protein obtained after gel filtration (A). Chromatography of 2.4 mg of a crude extract from K. lactis. (B) Chromatography of 3mg of partially purified extracts from Maxilact LX-5000. The profile of molecular weight standards used for calibration are shown. Absorbance values of standards are multiplied by 100. Fraction showing the peak of maximum lactase activity was estimated to correspond to a molecular weight of 250 kDa in the crude extract and 260 kDa in Maxilact LX-5000.

Article Snippet: In the first case, BALB/c mice were intraperitoneally immunized with 0.2 ml of a 1:1 (v/v) mixture of Freunds complete adjuvant (FCA) and PBS containing 50 mg of the K. lactis β-galactosidase (Maxilact LX-5000, Gist Brocades, France/The Netherlands).

Techniques: Activity Assay, Filtration, Chromatography, Purification, Molecular Weight

Journal: Biological Procedures Online

Article Title: Dealing with different methods for Kluyveromyces lactis β-galactosidase purification

doi: 10.1251/bpo4

Figure Lengend Snippet: Profiles of β-Galactosidase activity and protein obtained after ion-exchange chromatography. (A) Profile of 2.4 mg of a crude extract from K. lactis . (B) Chromatography of 0.6 mg of active fractions pooled from gel filtration of a crude yeast protein extract. (C) Chromatography of 2.4 mg protein of Maxilact LX-5000.

Article Snippet: In the first case, BALB/c mice were intraperitoneally immunized with 0.2 ml of a 1:1 (v/v) mixture of Freunds complete adjuvant (FCA) and PBS containing 50 mg of the K. lactis β-galactosidase (Maxilact LX-5000, Gist Brocades, France/The Netherlands).

Techniques: Activity Assay, Ion Exchange Chromatography, Chromatography, Filtration

Journal: Biological Procedures Online

Article Title: Dealing with different methods for Kluyveromyces lactis β-galactosidase purification

doi: 10.1251/bpo4

Figure Lengend Snippet: SDS-PAGE (left) and immunoblotting (right) of purified β-Galactosidase from K. lactis (A). β-Galactosidase is indicated by an arrow. Western-blot was incubated with mouse immune serum. Lane 1: 75 μg of a crude extract from K. lactis ; lane 2: Molecular weight markers; lane 3: 5 μg of β- galactosidase from K. lactis purified by affinity chromatography. (B) Electrophoresis on a non-denaturing gradient gel (5-15%) stained with ONPG and later Coomassie brilliant blue G250 (left) and with BNG (right). Bands with β-Galactosidase activity are indicated by higher arrows. Different forms of β-Galactosidase are shown by smaller arrows. Different concentrations of b-Galactosidase from Maxilact LX-5000 were loaded. Lane 1:20 μg; lane 2:40 μg; lane 3:80 μg.

Article Snippet: In the first case, BALB/c mice were intraperitoneally immunized with 0.2 ml of a 1:1 (v/v) mixture of Freunds complete adjuvant (FCA) and PBS containing 50 mg of the K. lactis β-galactosidase (Maxilact LX-5000, Gist Brocades, France/The Netherlands).

Techniques: SDS Page, Western Blot, Purification, Incubation, Molecular Weight, Affinity Chromatography, Electrophoresis, Staining, Activity Assay

Journal: Biological Procedures Online

Article Title: Dealing with different methods for Kluyveromyces lactis β-galactosidase purification

doi: 10.1251/bpo4

Figure Lengend Snippet: Results of cross-reactivity obtained in the ELISA performed with the specific anti K.lactis β-galactosidase antibodies prepared in this work (red bars) versus (A) a crude protein extract of the K.lactis β-galactosidase deficient strain MW-190-9B, (B) a crude protein extract of K.lactis NRRL-Y1140, (C) purified Maxilact LX-5000 β-galactosidase, (D) partially purified K. fragilis β-galactosidase, (E) Aspergillus niger β-galactosidase, (F) bovine liver β-galactosidase, (G) Escherichia coli β-galactosidase. Blue bars are controls of absence of reactivity when the serum of a non-immunized mouse was used instead of the specific anti K.lactis β-galactosidase antibodies. Yellow bars are controls of absence of reactivity when these primary antibodies are not added.

Article Snippet: In the first case, BALB/c mice were intraperitoneally immunized with 0.2 ml of a 1:1 (v/v) mixture of Freunds complete adjuvant (FCA) and PBS containing 50 mg of the K. lactis β-galactosidase (Maxilact LX-5000, Gist Brocades, France/The Netherlands).

Techniques: Enzyme-linked Immunosorbent Assay, Purification