Direkte immunologische Identifizierung von Proteinen nach elektrophoretischer Fraktionierung in Polyacrylamid-Mikrogradientengelen und Einfluß von Detergentien auf die Immunpräzipitation

Neuhoff, Volker; Mesecke, Senta

doi:10.1515/bchm2.1977.358.2.1623

Cited by 5 publications

(1 citation statement)

References 11 publications

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“…The production of capillary pipettes, repeatedly used in microelectrophoresis, was easy, provided a suitable burner was available [15,98]. Two-dimensional microimmunodiffusion [15,99,100] proved suitable for immunoprecipitation with volumes of less than 1 mL of antigen and antibody solutions, with the additional advantage of results that were already visible within minutes or hours, in contrast to days with the classical Ouchterlony test. Using the same approach, complex formation could be studied in other systems [15,101,102], e.g., for the characterization of a polymerase-template complex [15,103].…”

Section: Auxiliary Equipment For Microanalysismentioning

confidence: 99%

Microelectrophoresis and auxilary micromethods

Neuhoff

2000

Electrophoresis

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In this retrospect of approximately 30 years of work with micromethods, some of them developed in our own laboratory, their principles and application to different separation problems are described, such as one‐ and two‐dimensional microelectrophoresis in capillaries and microslab gels, isoelectric focusing in capillaries or microslab gels, microchromatography, microphotometry, and microfluorometry for qualitative and quantitative evaluation of separation patterns. In addition, some useful auxiliary methods are also described, e.g., a method for quantitative protein determination in a microliter volume when neither the volume nor the protein content in that volume are known, and methods for the determination of glycoproteins, amino acids, and sugars in the picomole range.

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Section: Auxiliary Equipment For Microanalysismentioning

confidence: 99%

Microelectrophoresis and auxilary micromethods

Neuhoff

2000

Electrophoresis

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Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels: A systematic analysis

1985

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A systematic analysis of protein staining in polyacrylamide gels with Coomassie Brilliant Blue (CBB) R-250 and G-250 using a high resolution densitometer allowing for quantitative measurements during staining and destaining has revealed that none of the published procedures allows quantitative measurements. Protein staining with CBB R-250 in methanol/water/acetic acid is poor, as is staining with CBB G-250 in trichloroacetic acid or perchloric acid, the latter two, however, allowing for a weak background staining. Consequently using the colloidal properties of the CBB dyes, stronger for G-250 than for R-250, it is possible to increase the sensitivity of protein staining to a detection limit of 0.7 ng bovine serum albuminlmm' gel. In addition, sensitive protein staining on a clear background is possible. Recipes are described (Section 3.11) for intensified protein staining with CBB G-250 using trichloroacetic acid or perchloric acid on a clear background. Optimal staining of proteins on a clear background can be performed with phosphoric acid and CBB G-250 in the presence of ammonium sulfate since under these conditions the colloidal state ofthe dye is optimized. Furthermore, conditions are described which allow the stable fixation ofthe protein-dye complex. Combining the optimized staining conditions with the stable fixation in 20 % ammonium sulfate allows for stepwise staining fore. g. detection of weak spots in addition to intense protein spots. The dependence of different staining procedures on gel thickness, gel concentration and compounds routinely used in polyacrylamide gel electrophoresis is also analysed. Calibration curves and application of the new procedure to biological material demonstrate its wide applicability. Convincing arguments for the colloidal properties of the CBB dyes are presented, formulating the rationale for intensified protein staining with CBB dyes in polyacrylamide gels without background staining. Abbreviations: BSA, bovine serum albumin; CBB, Coomassie Brilliant Blue; DMSO, dimethylsulfoxide; EDTA, ethylenediaminetetraacetic acid djsodium salt; MeOH, methanol; ~p -4 0 , id^^ p.40; PAG, polyacrylamide gel: PAGE, polyacrylamide gel electrophoresis; PCA, perchloric acid: PEG, polyethylene glycol; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; Tris, tris(hydroxymethy1)aminomethane 'C'VCH Verlagsgesellschaft mbH, D-6940 Weinheim. 1385 01 73-0835/85/0909-0427 $02.50/0

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