scholarly journals A mucus-secreting human colonic cancer cell line. Purification and partial characterization of the secreted mucins

1989 ◽  
Vol 258 (3) ◽  
pp. 793-799 ◽  
Author(s):  
J J Maoret ◽  
J Font ◽  
C Augeron ◽  
P Codogno ◽  
C Bauvy ◽  
...  
Keyword(s):  
Cell Line ◽  
Conditioned Medium ◽  
Gel Electrophoresis ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cancer Cell Line ◽  
Monosaccharide Composition ◽  
Polyacrylamide Gel ◽  
Antigenic Determinants ◽  
Normal Human

A stably differentiated clonal derivative (Cl.16E) of the human colonic adenocarcinoma cell line HT29 secretes in culture high-Mr glycoproteins that were purified from the serum-free conditioned medium by preparative SDS/polyacrylamide-gel electrophoresis. Analysis of the oligosaccharides released from the [3H]glucosamine-labelled high-Mr glycoproteins by alkaline-borohydride treatment showed that this material consisted of O-linked oligosaccharides (without any detectable N-linked oligosaccharides) that were eluted as three fractions from Bio-Gel P-6 columns. The main oligosaccharide fraction obtained after such treatment and desialylation was eluted together with a six-unit glucose polymer from a Bio-Gel P-4 column. Polyclonal antibodies were raised against the high-Mr glycoproteins, and in immunoblot analysis they reacted specifically with the high-Mr glycoproteins present in the conditioned medium. Furthermore, immunohistochemical staining of sections in paraffin wax revealed that these antibodies labelled normal human gastrointestinal mucins. We conclude that (1) the high-Mr glycoproteins prepared by SDS/polyacrylamide-gel electrophoresis are pure mucus glycoproteins on the basis of sensitivity to alkaline-borohydride treatment, monosaccharide composition and immunochemical and immunohistological findings, and (2) these mucins have antigenic determinants in common with the normal human gastrointestinal mucins.

scholarly journals Isolation from human chronic-lymphocytic-leukaemia cells of membrane glycoproteins associated with Fc-receptor functions. Physical parameters and production of polyclonal antibodies

1987 ◽  
Vol 245 (1) ◽  
pp. 75-83 ◽  
Author(s):  
G Gorini ◽  
G A Medgyesi ◽  
M Garavini ◽  
K J Dorrington ◽  
J Down
Keyword(s):  
Chronic Lymphocytic Leukaemia ◽  
Gel Electrophoresis ◽  
Lymphocytic Leukaemia ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fc Receptor ◽  
Polyacrylamide Gel ◽  
Two Dimensional ◽  
Membrane Glycoproteins

Two membrane glycoproteins that bound immune complexes and inhibited Fc-receptor- (FcR-)mediated functions in vitro were purified from human FcR+ chronic-lymphocytic-leukaemia cells. A multi-step purification was developed, consisting essentially in: (i) Tween 40 extraction of crude cell membranes; (ii) solubilization of membrane fragments by Renex-30; (iii) isolation of glycoproteins by affinity chromatography on Lens culinaris haemagglutinin-Sepharose; (iv) papain treatment of the eluted glycoproteins followed by gel-filtration chromatography; (v) purification by polyacrylamide-gel electrophoresis of two molecular species from the protein-size fraction enriched for immune-complex-binding activity. The two electrophoretically isolated components displayed apparent molecular masses of 70 and 45 kDa by SDS/polyacrylamide-gel electrophoresis and restricted charge heterogeneity by two-dimensional analysis. Two-dimensional peptide mapping revealed the presence of many peptides in common between the two proteins and the absence of a number of peptides in the 45 kDa component. These two polypeptides were used as immunogens to produce polyclonal antibodies that cross-reacted with both proteins and specifically inhibited FcR-mediated reactions in vitro. Furthermore, FcR-related components from detergent-extracted lysates of the human K562 and U937 cell lines or human placental membranes were revealed by the putative anti-FcR antibodies adsorbed on Protein A-Sepharose.

scholarly journals Benzene dioxygenase in Pseudomonas putida. Subunit composition and immuno-cross-reactivity with other aromatic dioxygenases

1987 ◽  
Vol 244 (3) ◽  
pp. 611-616 ◽  
Author(s):  
M Zamanian ◽  
J R Mason
Keyword(s):  
Pseudomonas Putida ◽  
Gel Electrophoresis ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cross Reactivity ◽  
Polyacrylamide Gel ◽  
Subunit Composition ◽  
Naphthalene Dioxygenase ◽  
Toluene Dioxygenase ◽  
Benzoate Dioxygenase

The terminal oxygenase component of benzene dioxygenase from Pseudomonas putida strain ML2 was shown to contain two subunits, of Mr 54,500 and 23,500, by SDS/polyacrylamide-gel electrophoresis. The native Mr of the terminal oxygenase was estimated to be 168,000 +/- 4000. Polyclonal antibodies raised against each of the subunits cross-reacted with two polypeptides in cell-free extracts from toluene-grown Pseudomonas putida strain N.C.I.B. 11767. The Mr values of these polypeptides were similar to those reported for the subunits from the terminal dioxygenase component of toluene dioxygenase. These polypeptides were present only when this strain was grown on toluene. No cross-reactivity was observed with subunits of the naphthalene dioxygenase or benzoate dioxygenase systems.

Assessing CMT cell line stability by two dimensional polyacrylamide gel electrophoresis and mass spectrometry based proteome analysis

2008 ◽  
Vol 71 (2) ◽  
pp. 160-167 ◽  
Author(s):  
Kelan Zhang ◽  
Krzysztof Wrzesinski ◽  
Stephen J. Fey ◽  
Peter Mose Larsen ◽  
Xumin Zhang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cell Line ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Proteome Analysis ◽  
Polyacrylamide Gel ◽  
Two Dimensional
Sign in / Sign up

Export Citation Format

Share Document