Mapping of polypeptide genes by two-dimensional gel electrophoresis of hybrid cell extracts

1982 ◽  
Vol 32 (1-4) ◽  
pp. 99-110 ◽  
Author(s):  
R.T. Taggart ◽  
U. Francke
Keyword(s):  
Gel Electrophoresis ◽  
Hybrid Cell ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Cell Extracts

scholarly journals Identification of Proteins Related to Nickel Homeostasis in Helicobater pylori by Immobilized Metal Affinity Chromatography and Two-Dimensional Gel Electrophoresis

2008 ◽  
Vol 2008 ◽  
pp. 1-6 ◽  
Author(s):  
Xuesong Sun ◽  
Ruiguang Ge ◽  
Jen-Fu Chiu ◽  
Hongzhe Sun ◽  
Qing-Yu He
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Interacting Proteins ◽  
Peptic Ulcers ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Iron Storage ◽  
Cell Extracts ◽  
Nickel Homeostasis ◽  
H Pylori

Helicobacter pylori (H. pylori) is a widespread human pathogen causing peptic ulcers and chronic gastritis. Maintaining nickel homeostasis is crucial for the establishment of H. pylori infection in humans. We used immobilized-nickel affinity chromatography to isolate Ni-related proteins from H. pylori cell extracts. Two-dimensional gel electrophoresis and mass spectrometry were employed to separate and identify twenty two Ni-interacting proteins in H. pylori. These Ni-interacting proteins can be classified into several general functional categories, including cellular processes (HspA, HspB, TsaA, and NapA), enzymes (Urease, Fumarase, GuaB, Cad, PPase, and DmpI), membrane-associated proteins (OM jhp1427 and HpaA), iron storage protein (Pfr), and hypothetical proteins (HP0271, HP jhp0216, HP jhp0301, HP0721, HP0614, and HP jhp0118). The implication of these proteins in nickel homeostasis is discussed.

scholarly journals Striated flagellar roots: isolation and partial characterization of a calcium-modulated contractile organelle.

1984 ◽  
Vol 99 (3) ◽  
pp. 962-970 ◽  
Author(s):  
J L Salisbury ◽  
A Baron ◽  
B Surek ◽  
M Melkonian
Keyword(s):  
Gel Electrophoresis ◽  
Calcium Binding ◽  
Protein Band ◽  
Blue Staining ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Flagellar Roots ◽  
Beta Form ◽  
Cell Extracts ◽  
Tetraselmis Striata

We report the isolation of striated flagellar roots from the Prasinophycean green alga Tetraselmis striata using sedimentation in gradients of sucrose and flotation on gradients of colloidal silica. PAGE in the presence of 0.1% SDS demonstrates that striated flagellar roots are composed of a number of polypeptides, the most predominant one being a protein of 20,000 Mr. The 20,000 Mr protein band represents approximately 63% of the Coomassie Brilliant Blue staining of gels of isolated flagellar roots. Two-dimensional gel electrophoresis (isoelectric focusing and SDS PAGE) resolves the major 20,000 Mr flagellar root protein into two components of nearly identical Mr, but of differing isoelectric points (i.e., pl's of 4.9 and 4.8), which we have designated 20,000-Mr-alpha and 20,000-Mr-beta, respectively. Densitometric scans of two-dimensional gels of cell extracts indicate that the 20,000-Mr-alpha and -beta polypeptides vary, in their stoichiometry, between 2:1 and 1:1. This variability appears to be related to the state of contraction or extension of the striated flagellar roots at the time of cell lysis. Incubation of cells with 32PO4 followed by analysis of cell extracts by two-dimensional gel electrophoresis and autoradiography reveals that the more acidic 20,000-Mr-beta component is phosphorylated and the 20,000-Mr-alpha component contains no detectable label. These results suggest that the 20,000-Mr-alpha component is converted to the more acidic 20,000-Mr-beta form by phosphorylation. Both the 20,000-Mr-alpha and -beta flagellar root components exhibit a calcium-induced reduction in relative electrophoretic mobilities in two-dimensional alkaline urea gels. Antiserum raised in rabbits against the 20,000-Mr protein binds to both the 20,000-Mr-alpha and 20,000-Mr-beta forms of the flagellar root protein when analyzed by electrophoretic immunoblot techniques. Indirect immunofluorescence on vegetative or interphase cells demonstrate that the antibodies bind to two cyclindrical organelles located in the anterior region of the cell. Immunocytochemical investigations at ultrastructural resolution using this antiserum and a colloidal gold-conjugated antirabbit-IgG reveals immunospecific labeling of striated flagellar roots and their extensions. We conclude that striated flagellar roots are simple ion-sensitive contractile organelles composed predominantly of a 20,000 Mr calcium-binding phosphoprotein, and that this protein is largely responsible for the motile behavior of these organelles.

scholarly journals Two-Dimensional Gel Electrophoresis Analyses of pH-Dependent Protein Expression in Facultatively AlkaliphilicBacillus pseudofirmus OF4 Lead to Characterization of an S-Layer Protein with a Role in Alkaliphily

2000 ◽  
Vol 182 (21) ◽  
pp. 5969-5981 ◽  
Author(s):  
Raymond Gilmour ◽  
Paul Messner ◽  
Arthur A. Guffanti ◽  
Rebecca Kent ◽  
Andrea Scheberl ◽  
...  
Keyword(s):  
Growth Rate ◽  
Gel Electrophoresis ◽  
Ph Regulation ◽  
Lag Phase ◽  
Two Dimensional ◽  
Wild Type ◽  
Ph Homeostasis ◽  
Two Dimensional Gel Electrophoresis ◽  
Cell Extracts ◽  
Ph Dependent

ABSTRACT The large majority of proteins of alkaliphilic Bacillus pseudofirmus OF4 grown at pH 7.5 and 10.5, as studied by two-dimensional gel electrophoresis analyses, did not exhibit significant pH-dependent variation. A new surface layer protein (SlpA) was identified in these studies. Although the prominence of some apparent breakdown products of SlpA in gels from pH 10.5-grown cells led to discovery of the alkaliphile S-layer, the largest and major SlpA forms were present in large amounts in gels from pH 7.5-grown cells as well. slpA RNA abundance was, moreover, unchanged by growth pH. SlpA was similar in size to homologues from nonalkaliphiles but contained fewer Arg and Lys residues. An slpA mutant strain (RG21) lacked an exterior S-layer that was identified in the wild type by electron microscopy. Electrophoretic analysis of whole-cell extracts further indicated the absence of a 90-kDa band in the mutant. This band was prominent in wild-type extracts from both pH 7.5- and 10.5-grown cells. The wild type grew with a shorter lag phase than RG21 at either pH 10.5 or 11 and under either Na+-replete or suboptimal Na+ concentrations. The extent of the adaptation deficit increased with pH elevation and suboptimal Na+. By contrast, the mutant grew with a shorter lag and faster growth rate than the wild type at pH 7.5 under Na+-replete and suboptimal Na+ conditions, respectively. Logarithmically growing cells of the two strains exhibited no significant differences in growth rate, cytoplasmic pH regulation, starch utilization, motility, Na+-dependent transport of α-aminoisobutyric acid, or H+-dependent synthesis of ATP. However, the capacity for Na+-dependent pH homeostasis was diminished in RG21 upon a sudden upward shift of external pH from 8.5 to 10.5. The energy cost of retaining the SlpA layer at near-neutral pH is apparently adverse, but the constitutive presence of SlpA enhances the capacity of the extremophile to adjust to high pH.

Diversity of Glycoprotein Deficiencies in Glanzmann’s Thrombasthenia

1985 ◽  
Vol 54 (03) ◽  
pp. 626-629 ◽  
Author(s):  
M Meyer ◽  
F H Herrmann
Keyword(s):  
High Resolution ◽  
Gel Electrophoresis ◽  
Type Ii ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Structural Variant ◽  
Glanzmann’S Thrombasthenia ◽  
Crossed Immunoelectrophoresis ◽  
Glanzmann's Thrombasthenia ◽  
Platelet Proteins

SummaryThe platelet proteins of 9 thrombasthenic patients from 7 families were analysed by high resolution two-dimensional gel electrophoresis (HR-2DE) and crossed immunoelectrophoresis (CIE). In 7 patients both glycoproteins (GPs) IIb and Ilia were absent or reduced to roughly the same extent. In two related patients only a trace of GP Ilb-IIIa complex was detected in CIE, but HR-2DE revealed a glycopeptide in the position of GP Ilia in an amount comparable to type II thrombasthenia. This GP Ilia-like component was neither recognized normally by anti-GP Ilb-IIIa antibodies nor labeled by surface iodination. In unreduced-reduced two-dimensional gel electrophoresis two components were observed in the region of GP Ilia. The assumption of a structural variant of GP Ilia in the two related patients is discussed.

Comparative Two-Dimensional Gel Electrophoresis of Trypanosoma cruzi Mammalian-Stage Forms in an Alkaline pH Range

2015 ◽  
Vol 22 (12) ◽  
pp. 1066-1075 ◽  
Author(s):  
Adriana Magalhães ◽  
Rayner Queiroz ◽  
Izabela Bastos ◽  
Jaime Santana ◽  
Marcelo Sousa ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Gel Electrophoresis ◽  
Two Dimensional ◽  
Alkaline Ph ◽  
Two Dimensional Gel Electrophoresis ◽  
Ph Range

Proteome Analysis of CD4+ T Cells Reveals Differentially Expressed Proteins in Infertile Polycystic Ovary Syndrome Patients

2020 ◽  
Vol 20 ◽  
Author(s):  
Fatemeh Nasri ◽  
Maryam Zare ◽  
Mehrnoosh Doroudchi ◽  
Behrouz Gharesi-Fard
Keyword(s):  
Mass Spectrometry ◽  
T Cells ◽  
T Lymphocytes ◽  
Gel Electrophoresis ◽  
Cd4 T Cells ◽  
Polycystic Ovary ◽  
Proteome Analysis ◽  
Two Dimensional ◽  
Ovary Syndrome ◽  
Two Dimensional Gel Electrophoresis

Background: Polycystic ovary syndrome (PCOS) is the most frequent endocrine disorder affecting 6–7% of premenopausal women. Recent studies revealed that the immune system especially CD4+ T helper cells are important in the context PCOS. Proteome analysis of CD4+ T lymphocytes can provide valuable information regarding the biology of these cells in the context of PCOS. Objective: To investigate immune dysregulation in CD4+ T lymphocytes at the protein level in the context of PCOS using two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS). Methods: In the present study, we applied two-dimensional gel electrophoresis / mass spectrometry to identify proteins differentially expressed by peripheral blood CD4+ T cells in ten PCOS women compared with ten healthy women. Western blot technique was used to confirm the identified proteins. Results: Despite the overall proteome similarities, there were significant differences in the expression of seven spots between two groups (P <0.05). Three proteins, namely phosphatidylethanolamine-binding protein 1, proteasome activator complex subunit 1 and triosephosphate isomerase 1 were successfully identified by Mass technique and confirmed by western blot. All characterized proteins were over-expressed in CD4+ T cells from patients compared to CD4+ T cells from controls (P <0.05). In-silico analysis suggested that the over-expressed proteins interact with other proteins involved in cellular metabolism especially glycolysis and ferroptosis pathway. Conclusion: These findings suggest that metabolic adjustments in CD4+ T lymphocytes, which is in favor of increased glycolysis and Th2 differentiation are important in the context of PCOS.

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