Comparison of yeast cell protein solubilization procedures for two-dimensional electrophoresis

Abstract Human erythrocyte lysate proteins were resolved into over 250 discrete spots by two-dimensional electrophoresis using isoelectric focusing in the first dimension and electrophoresis in the presence of sodium dodecyl sulfate, (SDS) in the second. The overwhelming excess of hemoglobin has made such analyses difficult in the past. However, with the ISO-DALT two-dimensional electrophoresis system, large numbers of red cell proteins can be mapped in the presence of hemoglobin. When hemoglobin and several other major proteins are removed by adsorption to DEAE-cellulose, additional minor components are seen, giving a total of over 275. With the use of purified preparations, the map positions of five cell enzymes or their subunits were determined: pyruvate kinase, catalase, glucose-6-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase, and carbonic anhydrase. The mapping techniques described complement and extend those traditionally used to find human red cell protein variants.

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Detergents and Chaotropes for Protein Solubilization Before Two-Dimensional Electrophoresis

Methods in Molecular Biology - Membrane Proteomics ◽

10.1007/978-1-60327-310-7_18 ◽

2009 ◽

pp. 259-267 ◽

Cited By ~ 24

Author(s):

Thierry Rabilloud

Keyword(s):

Two Dimensional ◽

Two Dimensional Electrophoresis ◽

Protein Solubilization ◽

Dimensional Electrophoresis

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Red cell proteins. I. Two-dimensional mapping of human erythrocyte lysate proteins

Blood ◽

10.1182/blood.v53.6.1121.bloodjournal5361121 ◽

1979 ◽

Vol 53 (6) ◽

pp. 1121-1132

Author(s):

JJ Edwards ◽

NG Anderson ◽

SL Nance ◽

NL Anderson

Keyword(s):

Human Erythrocyte ◽

Sodium Dodecyl ◽

Deae Cellulose ◽

Cell Protein ◽

Red Cell ◽

Two Dimensional ◽

Two Dimensional Electrophoresis ◽

Mapping Techniques ◽

Glucose 6 Phosphate Dehydrogenase ◽

Dimensional Electrophoresis

Human erythrocyte lysate proteins were resolved into over 250 discrete spots by two-dimensional electrophoresis using isoelectric focusing in the first dimension and electrophoresis in the presence of sodium dodecyl sulfate, (SDS) in the second. The overwhelming excess of hemoglobin has made such analyses difficult in the past. However, with the ISO-DALT two-dimensional electrophoresis system, large numbers of red cell proteins can be mapped in the presence of hemoglobin. When hemoglobin and several other major proteins are removed by adsorption to DEAE-cellulose, additional minor components are seen, giving a total of over 275. With the use of purified preparations, the map positions of five cell enzymes or their subunits were determined: pyruvate kinase, catalase, glucose-6-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase, and carbonic anhydrase. The mapping techniques described complement and extend those traditionally used to find human red cell protein variants.

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Use of Two-Dimensional Electrophoresis To Study Differential Protein Expression in Divercin V41-Resistant and Wild-Type Strains of Listeria monocytogenes

Applied and Environmental Microbiology ◽

10.1128/aem.66.10.4318-4324.2000 ◽

2000 ◽

Vol 66 (10) ◽

pp. 4318-4324 ◽

Cited By ~ 31

Author(s):

Frederique Duffes ◽

Paul Jenoe ◽

Patrick Boyaval

Keyword(s):

Transcriptional Regulation ◽

Listeria Monocytogenes ◽

Protein Expression ◽

Sensitive Strain ◽

Cell Protein ◽

Two Dimensional ◽

Protein Patterns ◽

Resistant Variant ◽

Two Dimensional Electrophoresis ◽

Dimensional Electrophoresis

ABSTRACT The use of bacteriocins from food-grade lactic acid bacteria to fight against the food-borne pathogen Listeria monocytogenes has been gaining interest. However, the emergence of resistant cells is frequently reported when Listeria is exposed to such antibacterials. A two-dimensional electrophoresis study of whole-cell protein expression of Listeria monocytogenes variants sensitive or resistant to the action of a bacteriocin produced by Carnobacterium divergens V41, divercin V41, is reported in this paper. The resistant variant obtained from the sensitive strain of L. monocytogenes P was also resistant to piscicocins V1 and SF668, but remained sensitive to nisin. Its growth rate was 50% less than the sensitive strain, and the MIC for it was 104 times higher. No reversion of the resistance was observed after 20 successive cultures in the absence of divercin V41. Comparison of the protein patterns by two-dimensional gel electrophoresis analysis showed clear differences. In the resistant variant pattern, at least nine spots had disappeared and eight new ones were observed. One of the newly synthesized proteins was identified as a flagellin of L. monocytogenes. Direct interaction between flagellin and divercin V41 was not evidenced. Intracellular synthesis of flagellin is probably an indirect effect of a modification in transcriptional regulation with widespread effects through a sigma factor. An intense protein, only present in the sensitive strain, was identified as a non-heme iron-binding ferritin displaying strong similarities to Dps proteins. Common modifications in the transcriptional regulation for these two proteins are discussed.

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Improved membrane protein solubilization and clean-up for optimum two-dimensional electrophoresis utilizing GLUT-1 as a classic integral membrane protein

Journal of Neuroscience Methods ◽

10.1016/j.jneumeth.2009.07.016 ◽

2009 ◽

Vol 184 (1) ◽

pp. 119-123 ◽

Cited By ~ 9

Author(s):

K. Devraj ◽

R. Geguchadze ◽

M.E. Klinger ◽

W.M. Freeman ◽

A. Mokashi ◽

...

Keyword(s):

Membrane Protein ◽

Integral Membrane Protein ◽

Two Dimensional ◽

Two Dimensional Electrophoresis ◽

Glut 1 ◽

Protein Solubilization ◽

Dimensional Electrophoresis

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Detergents and Chaotropes for Protein Solubilization before Two-Dimensional Electrophoresis

Plant Proteomics ◽

10.1385/1-59745-227-0:111 ◽

2006 ◽

pp. 111-120 ◽

Cited By ~ 2

Author(s):

Thierry Rabilloud ◽

Sylvie Luche ◽

Véronique Santoni ◽

Mireille Chevallet

Keyword(s):

Two Dimensional ◽

Two Dimensional Electrophoresis ◽

Protein Solubilization ◽

Dimensional Electrophoresis

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Sialic Acid Dependent Polypeptide Chain Heterogeneity of Human Fibrinogen Demonstrated by Two-Dimensional Electrophoresis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657116 ◽

1982 ◽

Vol 47 (01) ◽

pp. 019-021 ◽

Cited By ~ 13

Author(s):

Cemal Kuyas ◽

André Haeberli ◽

P Werner Straub

Keyword(s):

Sialic Acid ◽

Polypeptide Chain ◽

Two Dimensional ◽

Charge Heterogeneity ◽

Human Fibrinogen ◽

Two Dimensional Electrophoresis ◽

Isoelectric Points ◽

Polypeptide Chains ◽

Dimensional Electrophoresis ◽

Chain Type

SummaryHuman fibrinogen was compared with asialofibrinogen by two-dimensional electrophoresis to evaluate the contribution of sialic acid to the heterogeneity of the γ- and Bβ-polypeptide chains.Reduced fibrinogen showed three major variants for both the γ- and Bβ-chains. In addition two minor γ-bands with a more acidic isoelectric point than the normal γ-chains were observed. Electrophoresis in the second dimension (SDS) suggests that these most acidic bands are γ-chain-variants with a higher molecular weight. In asialofibrinogen only two predominant variants with more alkaline isoelectric points were present in each chain type.It is concluded that enzymatic removal of sialic acid partially reduces the heterogeneity of the γ- and Bβ-polypeptide chains of human fibrinogen, but additional sources producing charge heterogeneity must be sought.

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