Electrophoresis staining solutions

After completion of electrophoresis, the gel was stained with Coomassie blue. To prepare the staining solution, 40 mg Coomassie blue R-250 was dissolved in 25 ml isopropanol and 10 ml glacial acetic acid. The solution was filtered and the volume was increased to 100 ml with distilled water. The gel was placed in staining solution for 1 h, followed by washing with 10% acetic acid/2.5% isopropanol. 

Run the slab gel in a vertical dimension. The current should be set at 25-30 mA per slab gel. Allow the electrophoresis to proceed until the tracking dye is at the bottom of the gel. Turn the power off and remove the gels from the buffer chambers. Carefully separate the sandwiched plates with a thin spatula and transfer the gel in one piece to a tray containing staining solution. 

The slab gel used to obtain the data given in Table 4.3 was also prepared according to the method of Swank and Munkres (ref. 328), except the samples were run for 15 hr at a constant current of 15 mA/gel, followed by 3 hr at 50 mA/gel, and the protein-staining solution was applied for a period of 12 hours after electrophoresis. 

Staining. A variety of dyes which bind to proteins have found use in electrophoresis(4). One of the most sensitive and frequently used dyes is Coomassie Blue. Usually the staining procedure involves a "fixative", such as trichloroacetic acid, which precipitates proteins and prevents their diffusion out of the gel. Gels are soaked in the staining solution, at which point the entire gel is stained uniformly and unbound dye must be removed in a... 

The sample bands are not usually visible during electrophoresis (unless intrinsically coloured or fluorescent), but are normally visualized afterwards by immersing the gel in specific staining solutions. For DNA samples, intercalating fluorescent dyes such as ethidium bromide (see Section 2.4) are used, whereas proteins can be visualized using histological stains such as Coomassie blue or more sensitive silver stains. 

SDS-PAGE gels (10%), electrophoresis buffers, staining solution, and... 

Figure 3.11. Detection by polyacrylamide gel-electrophoresis of superoxide dismutase (A) and peroxidase (B) activities in crude extracts of P. coccoides (1), P. shermanii (2) and P. globosum (3). A gel stained for superoxide dismutase activity according to Beauchamp and Fridovich (1971), Stain solutions contained no KCN. B gel stained for peroxidase activity according to Gregory and Fridovich (1974). From Kraeva and Vorobjeva (1981b).

Figure 3. Line drawing of cellulose acetate gel electrophoresis of partially purified PP-ribose-P synthetase from 2 normal individuals and from patient T.B. The gel developed without PP-ribose-P in the staining solution represents a control gel. PP-ribose-P synthetase from patient T.B. migrates further than the enzyme from controls.

Following electrophoresis, rinse the gel with water and transfer it carefully to a container with Coomassie Blue Staining solution. 

Gel electrophoresis provides a simple method for separating complex protein mixtures. Because proteins are visualized using stains that may not be linearly incorporated in the gel, the intensity of the stained bands may be poorly correlated with the amount of protein. For this reason, gel electrophoresis is at best a semiquantitative technique capable of generating relative purity results. In CE, separations are commonly performed in free solution, i.e., in the absence of any support such as gel matrices. This allows the replacement of the capillary s content in between analyses and therefore the automation of the process. The use of UV-transparent fused-silica capillaries enables direct on-line optical detection of focused protein zones, eliminating the requirement for sample staining. The detection systems available to CE provide true quantitative capabilities. 

Based on its nature (aqueous solutions, physiological conditions, well-investigated labeling, and staining reactions) and the historical transition from slab-gel electrophoresis to CE, the main targets are biological and bioequivalent samples such as proteins, peptides, polynucleotides, oligonucleotides, and carbohydrates. 

Visual examination of crystals using a light microscope does not indicate whether the crystals consist of only the protein or the protein-DNA complex. Therefore, the crystals are washed free of any uncrystallized DNA and protein several times with a solution containing the precipitant and any additives, etc. at the concentration and pH used for growing crystals (mother liquor). Finally, the crystals are separated from the mother liquor by microcentrifugation, dissolved in a suitable buffer, and analysed biochemically. The protein content is determined by SDS polyacrylamide gel electrophoresis, the protein concentration by BIO-RAD assay, and amino acid composition by mass-spectroscopy. The DNA can be detected by staining the gel with ethidium bromide or methylene blue (Jordan et al., 1985), whereas... 

Place test and reference solutions, contained in covered test-tubes, in a waterbath for 2 min. Apply 10 ul of reference solution (f) and 50 pX of each of the other solutions to the stacking gel wells. Perform the electrophoresis under the conditions recommended by the manufacturer of the equipment. Detect proteins in the gel by silver staining. 

A variety of methods are available to detect proteins separated by electrophoresis or to measure the concentration of total protein in a solution. These methods are normally based on the binding of a dye to one of the amino acids in protein, or a color reaction with an amino acid side chain. The most commonly used stains for protein detection on gels are Coomassie Brilliant Blue (98) and silver stain (99,100). These methods detect any protein residues, either in solution or on an electrophoresis gel. Their main requirement is sensitivity, not specificity. New, more sensitive dyes are being developed for the proteomic analysis of protein structure and sequence, for example Ruby Red (101). 

After electrophoresis, transfer the gel into ethidium bromide solution (50 pi stock solution in 1 1 of tap water) and stain for 5 -10 min. Transfer the gel onto a transilluminator and visualise the DNA with UV light, taking care to protect the skin and eyes against UV radiation. Document the result with a standard photograph or digital image analyser. 

I Gel electrophoresis is the best way to analyze mix-Vr-j tures and assess purity. Gel electrophoresis separates proteins according to their size and their charge. It is almost always performed in aqueous solution supported by a gel system. The gel is a loosely cross-linked network that functions to stabilize the protein boundaries between the protein and the solvent, both during and after electrophoresis, so that they may be stained or otherwise manipulated. 

Protein concentrations were determined according to the method of Lowry et al. (30). Electrophoresis of proteins in polyacrylamide gels was carried out at 4°C, using the discontinuous buffer system No. 1 described by Maurer (31) and modified by Emert et al. (1). Protein was stained with 0.1% Coomassie Brilliant Blue R250 in a water-acetic acid-methanol (45 10 45) solution. Carbohydrates were stained with the periodic acid-Schiff (PAS) reagent using the method described by Lang (32). 

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