scholarly journals Antibody-affinity purification of novel α-l-fucosidase from mouse liver

1987 ◽  
Vol 245 (2) ◽  
pp. 589-593 ◽  
Author(s):  
L D Laury-Kleintop ◽  
I Damjanov ◽  
J A Alhadeff
Keyword(s):  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Human Liver ◽  
Mouse Liver ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Antibody Affinity ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Sepharose 4B

Previous studies have documented the presence of a novel alpha-L-fucosidase in mouse liver that contains unique basic isoelectric forms and that is antigenically similar to, but not identical with, human liver alpha-L-fucosidase [Laury-Kleintop, Damjanov & Alhadeff (1985) Biochem. J. 230, 75-82]. In the present investigation, mouse liver alpha-L-fucosidase was purified approx. 26,500-fold in 10% overall yield by antibody-affinity chromatography with the IgG fraction of goat anti-(human alpha-L-fucosidase) antibody coupled to Sepharose 4B. Native polyacrylamide-gel electrophoresis and SDS/polyacrylamide-gel electrophoresis indicated that the mouse fucosidase is highly purified if not homogeneous. Isoelectric focusing demonstrated that all enzymic forms found in crude mouse liver supernatant fluids were purified by the antibody-affinity procedure.

scholarly journals Affinity purification and some molecular properties of human liver alkaline phosphatase

1976 ◽  
Vol 155 (3) ◽  
pp. 653-660 ◽  
Author(s):  
J M Trépanier ◽  
L E Seargeant ◽  
R A Stinson
Keyword(s):  
Alkaline Phosphatase ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Human Liver ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Neuraminidase Treatment

Alkaline phosphatase from human liver was purified to homogeneity. The purification procedure included solubilization with butanol, fractionation with acetone, and chromatography on concanavalin A-Sepharose, DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex. Purity was established by standard and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The isoelectric point of the protein was determined to be 4.0. Sephadex-gel filtration gave a mol.wt. of 146000, although a higher value was obtained in the presence of 100mM-NaC1. The subunit mol.wt. 76700, was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Neuraminidase treatment resulted in two enzyme-activity bands on isoelectric-focused gels with isoelectric points of 6.6 and 6.8. The desialylated enzyme gave only one protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with a subunit molecular weight indistinguishable from that of the non-neuraminidase-treated protein. The desialylated enzyme was more readily denatured by sodium dodecyl sulphate in the presence of mercaptoethanol than was the native enzyme.

Ultrastructural localization of specific nucleoprotein fractions

1978 ◽  
Vol 36 (2) ◽  
pp. 204-205
Author(s):  
G. L. Brown
Keyword(s):  
Gel Electrophoresis ◽  
Mouse Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ultrastructural Localization ◽  
Polyacrylamide Gel ◽  
Acid Extraction ◽  
Acid Solutions ◽  
Low Salt ◽  
Liver Nuclei ◽  
Phosphate Groups

Bismuth (Bi) stains nucleoproteins (NPs) by interacting with available amino and primary phosphate groups. These two staining mechanisms are distinguishable by glutaraldehyde crosslinking (Fig. 1,2).Isolated mouse liver nuclei, extracted with salt and acid solutions, fixed in either formaldehyde (form.) or gl utaraldehyde (glut.) and stained with Bi, were viewed to determine the effect of the extractions on Bi stainina. Solubilized NPs were analyzed by SDS-polyacrylamide gel electrophoresis.Extraction with 0.14 M salt does not change the Bi staining characteristics (Fig. 3). 0.34 M salt reduces nucleolar (Nu) staining but has no effect on interchromatinic (IC) staining (Fig. 4). Proteins responsible for Nu and glut.- insensitive IC staining are removed when nuclei are extracted with 0.6 M salt (Fig. 5, 6). Low salt and acid extraction prevents Bi-Nu staining but has no effect on IC staining (Fig. 7). When nuclei are extracted with 0.6 M salt followed by low salt and acid, all Bi-staining components are removed (Fig. 8).

scholarly journals Affinity purification and subunit structures of β-N-acetylhexosaminidases A and B from boar epididymis

1984 ◽  
Vol 219 (3) ◽  
pp. 1009-1015 ◽  
Author(s):  
H C Parkes ◽  
J L Stirling ◽  
P Calvo
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Gradient Gel Electrophoresis ◽  
Sepharose 4B ◽  
Electrophoretic Transfer ◽  
Peptide Maps

beta-N-Acetylhexosaminidase from boar epididymis was separated into two forms, A and B, on DEAE-cellulose. Both these forms were excluded from Sepharose S-200 and had apparent Mr values of 510 000 on gradient gel electrophoresis under non-denaturing conditions. Affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylam ine coupled to CNBr-activated Sepharose 4B was used to separate and purify beta-N-acetylhexosaminidases A and B that had specific activities of 115 and 380 mumol/min per mg of protein respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of denatured beta-N-acetylhexosaminidase A gave a single major component of Mr 67 000. beta-N-Acetylhexosaminidase B also had this component, and in addition had polypeptides of Mr 29 000 and 26 000. All these polypeptides were glycosylated. Antiserum to the B form precipitated form A from solution and reacted with the 67 000-Mr component or form A after electrophoretic transfer from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose sheets. The 67 000-Mr components of forms A and B yielded identical peptide maps when digested with Staphylococcus aureus V8 proteinase, and the 29 000-Mr and 26 000-Mr components in form B may be related to the 67 000-Mr polypeptide.

scholarly journals Common epitopes of bovine lens multicatalytic-proteinase-complex subunits

1989 ◽  
Vol 257 (1) ◽  
pp. 265-269 ◽  
Author(s):  
B J Wagner ◽  
J W Margolis
Keyword(s):  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyclonal Antiserum ◽  
Antibody Affinity ◽  
Multicatalytic Proteinase ◽  
Bovine Lens ◽  
The Common ◽  
Multicatalytic Proteinase Complex ◽  
Monospecific Antibodies

Component polypeptides of both the bovine lens and pituitary multicatalytic proteinase complexes demonstrate different immunoreactivities with a polyclonal antiserum raised against the purified pituitary enzyme. Four (Mr 24000, 26000, 34000 and 38000) of eight bands that have been resolved by SDS/polyacrylamide-gel electrophoresis are stained in immunoblot experiments. Monospecific antibodies obtained from this antiserum by affinity purification from the 38000- and 34000-Mr bands of the lens enzyme bound equally well to either band, but showed little or no binding to the 26000- and 24000-Mr bands upon immunoblotting. Antibody affinity-purified from the 24000-Mr band showed comparable binding to the 24000-, 34000- or 38000-Mr band. One explanation of these results is that the 24000-Mr polypeptide is derived from the higher-Mr polypeptide(s) and has lost some of the common immunodeterminants.

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