Quantitative Aspects of Protein Staining by Coomassie Blue Dyes and Silver Staining

Baker

1988

Forty-seven subjects diagnosed as having inhalant allergies to fungi were tested for allergic sensitivity to bakers’ yeast. Skin prick tests with yeast extract showed that 35 subjects responded with wheal reactions that were at least 3 mm while 32 subjects were regarded as clearly RAST-positive to bakers’ yeast antigens. Skin and RAST testing with purified enolase from bakers’ yeast and comparisons with the whole yeast extract showed that the enzyme is a major allergenic component of the extract. This conclusion was supported by results of electroblotting studies. RAST inhibition experiments demonstrated allergenic cross-reactivity between bakers’ yeast, bakers’ yeast enolase and Candida albicans.

“…Gradient gels were stained with Coomassie brilliant blue G250 (Sigma Chemical Co., St. Louis, Mo.) by the method of Neuhoff and Stamm [14].…”

Section: Gel Electrophoresismentioning

confidence: 99%

Section: Skin Prick Test and Rast Findingsmentioning

confidence: 99%

Inhalant Allergies to Fungi: Reactions to Bakers’ Yeast (Saccharomyces cerevisiae) and Identification of Bakers’ Yeast Enolase as an Important Allergen

Baker

1988

“…PAGs were stained with Coomassie brilliant blue G250 (CBB: Sigma Chemical Co.,) using the enhancing method described by Neuhoff and Stamm [1984], NC transfers were stained with amido black.…”

Section: Protein Stainingmentioning

confidence: 99%

A Re-examination of Ryegrass (Lolium perenne) Pollen Allergens

Ford

1986

Electroblotting of crude ryegrass pollen extracts and purified group I, II and III allergens identified 14 IgE-binding components, 8 of which were previously unrecognized. In addition to allergen groups I, II and III, which are already regarded as clinically important, and on the basis of the frequencies and intensities of IgE binding with sera from 42 ryegrass pollen-allergic patients, proteins with molecular weights (MWs) of 60, 32, 30 and 28 kD were identified as allergens of possible major clinical importance. Six other pollen components with MWs ranging from 23 to 80 kD and which reacted with IgE antibodies in the sera of 33–50% of patients, should also be viewed as proteins with potential clinical relevance for at least a proportion of the patients. The electrophoretic separation patterns of ryegrass pollen extracts in both alkaline and acid gels and IgE-probed membrane transfers produced in this study should serve as useful reference patterns for standardization purposes. In addition, the identification of the complete allergen recognition pattern by individual patients will permit safer and more effective diagnosis and therapy of ryegrass pollen sensitivities.

“…Protein Staining. Gels were stained with Coomassie Brilliant Blue G-250 (CBB; Sigma Chemical Co.) using the enhanced stain ing method described by Neuhoff and Stamm [1984], NC transfers were stained with amido black.…”

Section: Gradient Gel Electrophoresismentioning

confidence: 99%

Identification of Parietaria judaica Pollen Allergens

Ford

Geraci

et al. 1986

Parietaria judaica pollen allergens, fractionated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, were identified using 52 sera collected in Australia and Sicily from P. judaica pollen-allergic patients. IgE-binding pollen components transferred to nitrocellulose were detected by reaction with ¹²⁵I-anti-human IgE and autoradiography. Nine pollen components, ranging in molecular weight (MW) from approximately 10,000 to 80,000 daltons, bound IgE antibodies but the two fastest migrating components sometimes each separated into two very closely migrating bands. The faster of the two components exhibiting doublet formation (MW approximately 10,000 daltons) showed by far the highest frequency of IgE binding, being recognised by 50 of the 52 sera examined. Although patients’ IgE reaction patterns to P. judaica allergens were heterogeneous, the degree of heterogeneity was much less than that observed with house dust mite and other pollen extracts studied by electrophoretic transfer analysis. Results with gradient gel-nitrocellulose transfer experiments which showed no IgE-binding components with MWs less than 70,000 daltons, and comparisons of our electroblotting results with crossed radioimmunoelectrophoresis results of others, suggested that the doublet proteins with MWs of approximately 10,000 probably bind to higher MW proteins in P. judaica pollen extracts.