Detection of biomolecules in electrophoresis gels with salts of imidazole and zinc II: A decade of research

Abstract

The proven ability of gel electrophoresis to simultaneously resolve, in a single experiment, many components from complex biological samples, has determined its preference over a variety of well-established chromatographic methods. Therefore, procedures placed at the interface between gel separation and microanalysis have earned increasing significance with respect to the overall success of the microanalytical strategy. The first of these procedures is the detection technique. The most important requirement for compatibility with further analysis or bioapplications is that the staining method does not compromise the chemical integrity and the biological properties of micropurified biomolecules. Procedures for negative detection of proteins with metal salts that have been proven to comply with this condition have been known for about 15 years. Only recently have these procedures been extended to the field of nucleic acids and lipopolysaccharides. The focus of this review is to chronicle the development and current status of the negative or reverse stain procedure based on the in-gel reaction of imidazole with zinc salts and its applications forthe micropurification and analysis of unmodified proteins, nucleic acids and bacterial lipopolysaccharides. We highlight the common aspects in the detection of the three types of biomolecules, and their applications to structural and biological analyses. Emphasis is given on the mechanism underlying imidazole-zinc staining, as it contributes to a deeper understanding of a general detection mechanism with metal salts. Finally, we discuss the latest applications of the techniques in proteomics and their possible impact on the characterization of gel-separated single components from complex lipopolysaccharides.

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

“…The consensus for zinc-imidazole-stained gels Yes [20], [73], [157,158] 500 ng A. thaliana total protein SR, MeOH/HAc fixation, stain from 3 h to overnight, destain in MeOH/HAc, water washed. was also to take images, against a black background in this case, usually using a Polaroid camera or an automated imager, and follow with analysis [20,100,101,104,107,108,243]. Fluorescence posed a different challenge in terms of detection given the specific excitation and emission requirements of different fluorophores.…”

Microchip electrophoretic separation for the fast diagnosis of Anaplasma phagocytophilum infection in cattle

“…To evaluate the ME system for its ability to detect the 16S rRNA gene, results from the PCR products were compared with traditional slab gel electrophoresis. In conventional slab gel electrophoresis, the LOD for a typical DNA sample is in the nanogram range ( Â 10 À9 g) per mL, similar to 8-10 ng/mL for typical Coomassie Blue strain and 30-100 ng/mL for colloidal Coomassie Blue strain [15,16]. The ME system uses a laser-induced fluorescence detector, thus linear regression equations for 900 and 1000 bp DNA molecules in the range of 0.45-9 and 0.2-10 ng/mL were y 5 0.0008x 10.0131 (R 5 0.9841) and y 5 0.0029x10.0193 (R 5 0.9938), respectively.…”

An optimized procedure for detection of proteins on carrier ampholyte isoelectric focusing and immobilized pH gradient gels with imidazole and zinc salts: Its application to the identification of isoelectric focusing separated isoforms by in-gel proteolysis and mass spectrometry analysis

“…Imidazole-zinc detection on SDS-gels was done according to published procedures [1,2,9]. Imidazole-zinc detection on native or 8 M urea CA-IEF slab gels and IPG strips: All steps were done in a transparent plastic tray under continuous gentle agitation.…”

“…Due to its sensitivity and reproducibility, imidazole-zinc negative staining has been successfully used as detection technique for micropreparative purposes ( [1] and citations therein); its application in proteome projects has been reviewed [2]. Recently, high sequence coverage (90-97%) was described by combining protein detection using imidazole-zinc gel staining with an improved digestion protocol on gel microparticles [1,3].…”