Protein blotting: Principles and applications

The isolation and biochemical characterization of the extracellular form of a cellulose-binding factor (CBF) from Clostridium thermocellum is described. The CBF was isolated from the culture supernatant by a two-step procedure which included affinity chromatography on cellulose and gel filtration on Sepharose 4B. The isolated CBF was homogeneous as determined by immunoelectrophoresis, polyacrylamide gel electrophoresis, gel filtration, and analytical ultracentrifugation analysis. The CBF was found to form a complex which exhibited a molecular weight estimated at 2.1 million. Electron microscopic analysis of negatively stained preparations of the isolated CBF revealed a particulate, multisubunit entity of complicated quaternary structure. The molecule appeared to be about 18 nm in size. Although urea failed to break the complex into its component parts, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate resolved the CBF complex into 14 polypeptide bands. Immunoprecipitation experiments confirmed that these polypeptides indeed formed part of the same complex. Interestingly, by using the whole-cell immunization procedure described in the accompanying article (Bayer et al., J. Bacteriol., 156:818-827, 1983) only one CBF subunit (Mr = 210,000) was found to be antigenically active. By using a gel-overlay assay technique, at least eight of the remaining CBF-associated polypeptide components were shown to exhibit cellulolytic activity. The results are consistent with the contention that the CBF comprises a discrete, multisubunit complex or group of closely related complexes which exhibit separate antigenic and multiple cellulase activities in addition to the property of cellulose binding. It appears that the CBF is not only responsible for the adherence of the cells to cellulose but also constitutes a major part of the cellulolytic apparatus of this organism.

Characterization of a cellulose-binding, cellulase-containing complex in Clostridium thermocellum.

Detection of chitinase activity after polyacrylamide gel electrophoresis

We have developed a simple and reproducible technique that we have termed “track dilution” for enumerating viable bacteria, which reduces time and materials required compared to standard agar plate colony counting methods. Many microbiological applications require an accurate determination of bacterial numbers prior to inoculation into, or subsequent to their isolation from, experimental models and systems. For example, our experimental infection models require injection of predetermined numbers of viable colony-forming units (cfu) (1,4). The model further requires careful monitoring of bacterial growth rates at specific time points during infection. In our models of rodent gastrointestinal ecology (3), bacterial concentration must be determined from extraordinarily large numbers of fecal samples. The most commonly used technique for enumerating bacterial cfu involves serial dilution of samples followed by spreading 50–100 μL of each dilution onto agar media. Subsequent to incubation, colonies are counted and bacterial concentrations in the original sample estimated. This technique, though relatively accurate, has some drawbacks, including time required and a considerable expenditure of disposable plastics and media. To meet criteria for statistical accuracy of bacterial numbers in a given specimen samples are usually plated in triplicate, and cfu counted only from plates yielding between 20 and 200 visible colonies (2). Furthermore, since the actual number of bacteria present in a specimen at the completion of an experiment is unknown, samples must frequently be plated from dilutions ranging from 10-1 to 10-7. The result of such exhaustive measures is that most plated samples will yield either too many colonies to count or no colonies at all. For example, an overnight bacterial broth culture containing 109 cfu/mL, serially diluted 10fold over a range of 10-1 to 10-7 and plated (100 μL per plate) in triplicate, will yield only three agar plates with countable and statistically valid numbers of cfu; the remaining 18 plates must be discarded. To simplify bacterial enumeration and reduce time and material expenditures, we have developed a track-dilution technique whereby six 10-fold serial dilutions of a sample containing an unknown quantity of bacteria are plated onto a single agar plate. We compared track dilution with the standard spreadplate method by: (i) recording time required to process samples, (ii) recording materials expended and (iii) statistically analyzing the data for accuracy and reproducibility. Enterococcus faecalis and Staphylococcus aureus were propagated in brain-heart infusion (BHI) broth (Difco Laboratories, Detroit, MI, USA) overnight at 37°C without aeration. Bacillus subtilis and Escherichia coli DH5α (Life Technologies, Gaithersburg, MD, USA) were propagated in LB overnight at 37°C with aeration. Cultures were serially diluted 10-fold by sequential transfer of 100 μL into 900 μL phosphate-buffered saline (PBS). Experiments were performed in triplicate to allow statistical comparisons between the two techniques. For conventional spread-plate counting, cultures were diluted by a factor of 10-7, from which 100-μL samples were spread onto the surface of prewarmed BHI 1.5% agar plates (100× 15-mm round plates; Fisher Scientific, Pittsburgh, PA, USA) using a sterile bent-glass rod and an inoculating turntable (Fisher Scientific). Plates were incubated upright for ap-

https://www.future-science.com/doi/pdf/10.2144/97234bm22

Simplified agar plate method for quantifying viable bacteria

A sensitive method for detection of calmodulin-dependent protein kinase II activity in sodium dodecyl sulfate-polyacrylamide gel

http://www.jbc.org/content/255/15/7467.full.pdf

S.S. Springhorn

Papers

Renaturation of enzymes after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.