Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.Several improvements of the original Coomassie protocol¹ have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution², and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol³. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff''s colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol⁴. The novel aluminum-based staining in Kang''s study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.Here, we demonstrate application of Kang''s protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.Download video file.^{(128M, mp4)}     

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.Download video file.^{(88M, mp4)}     

Bestimmung von Diffusionskoeffizienten biologischer und anderer Lösungen mittels der Methode des fallenden Films

Zusammenfassung Es werden theoretische Überlegungen über die Messung von Diffusionskoeffizienten an physiologischen Membranen durchgeführt.Ein neu konstruiertes Gerät zur schnellen Bestimmung des Mittelwertes ¯D des Diffusions -koeffizienten mit Hilfe der Methode desfallenden Films wird beschrieben.

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Determination of diffusion coefficients of biologic and other solutions by the falling- film method

Summary Theoretical considerations about the measurement of diffusion coefficients in physiological membranes are given.A new method and a new instrument for the determination of the mean values ¯D of diffusion coefficients by means of a falling blood film are described.

     

Einfluß extracellulärer Konzentrationen auf die spezifische Wachstumsrate von Mikroorganismen

The influence of extracellular concentrations of hydrogen ion and C-substrates on the specific growth rate of different microorganisms is investigated. A general relationship in the form can be used to describe the inhibition effect of H^Plus;- or substrate concentration (c_i) on the growth rate. Different examples are demonstrated and the adequate specific constants K₁ and K₂ calculated.     

Convergence des référentiels de qualité et implications pour la fonction technique biomédicale

Risk management in the hospital, which is one of the referentiels of the ANAES accreditation manual, may be considered on two levels. Firstly, risk management may be approached globally, in the same way as it is tackled in the accreditation process. Secondly, risk management may be more definite. A specific risk chosen in accordance with the priorities of a particular plan may be dealt with individually. In this respect, the tranfusion process allows the risk management method to be tested and developed.

Biotechnologische Produktion mikrobieller Wirkstoffe: ein Beispiel für die Nutzung des Adaptationspotentials von Mikroorganismen

The role of the microbial secondary metabolism in the cytodifferentiation of producer strains and in the ecological system is reviewed. The production of bioactive metabolites such as antibiotics seems to play a role in long-term strategies of adaptation to environmental changes. This is reflected by characteristic features of regulation of the secondary metabolism such as catabolite repression, lack of uniform regulatory mechanisms and limited specificity of enzymes involved. The examples given may show that advantages have been taken from the knowledge of special regulatory features with regard to strain improvement and the designing of the metabolite spectrum.