Coomassie Brilliant Blue R-250 Staining Research Articles

Mixed-dye staining method for protein detection in polyacrylamide gel electrophoresis using calconcarboxylic acid and rhodamine B.

We have developed a new mixed-dye protein staining method that is simple, rapid, and sensitive. A freshly prepared mixture of calconcarboxylic acid (NN, 0.02%) and rhodamine B (RB, 0.04%) in 40% methanol/7% acetic acid, was used as a staining solution. RB acts as an auxiliary agent to inhibit the binding of NN to the gel matrix, reducing the background staining and therefore enhancing the protein staining by NN. This mixed-dye staining method reduces the total staining and destaining time to less than an hour, and increases the sensitivity to 25 ng of bovine serum albumin, which is greater than the 100 ng sensitivity limit of Coomassie Brilliant Blue R-250 (CBBR) staining.

Molecular characterization of cellular proteins from colorectal tumors.

Recent evidence has suggested that progressive stages of colorectal tumorigenesis can be defined by a sequence of genetic events characterized by altered expression of certain genes and the appearance of cancer-specific proteins. Although the significance of these events is still not clear, expression of cancer-specific protein components may be directly involved in the neoplastic transformation. The purpose of the present study was to compare molecular characteristics of cellular proteins from human colorectal tumors and normal colonic mucosa. Normal mucosa and colorectal tumors from 18 patients were fractionated by a differential centrifugation scheme into four cellular fractions, i.e., nuclear, mitochondrial (10P), microsomal (100P) and cytosolic (100S). The proteins of these fractions from normal and tumorigenic mucosa were analyzed by one-dimensional polyacrylamide gel electrophoresis followed by Coomassie brilliant blue R-250 and silver nitrate staining. Nuclear proteins from normal and neoplastic tissues which had revealed the most significant diversities were further characterized by two-dimensional gel electrophoresis. Electrophoretically cancer-specific nuclear proteins in the molecular mass zone 35-40 kDa were used as immunogen to produce rabbit polyclonal antibodies. Electrophoretic analysis by one-dimensional gel electrophoresis showed clear differences in molecular characteristics of cellular proteins between normal and tumorigenic mucosa, especially among nuclear fractions. The latter were also confirmed by their two-dimensional electrophoresis results. Rabbit antibodies raised against electrophoretically specific nuclear proteins characterized by molecular mass of 35-40 kDa cross-reacted with 36 kDa polypeptide in 15 of 18 (83.3%) studied nuclear fractions of colorectal tumors but not with any normal mucosa. In some cases, nuclear cancer-associated components of 38 and 40 kDa were also recognized by these antibodies. During colorectal carcinogenesis, specific expression of several nuclear proteins takes place. One of them, the polypeptide of 36 kDa not found in normal colonic epithelium, was shared by over 83% of the studied carcinomas despite variations in detailed cancer properties. This particular nuclear protein may be considered as a potential marker for the colon malignancy.

Synthesis of Nα-[3H]Acetyl-L-lysine Chloromethyl Ketone and Its Use in the Fluorographic Detection of Proteases

Tritiated Nα-acetyl-L-lysine chloromethyl ketone (ALCK) was synthesized on a laboratory scale for use as an active-site-directed affinity label in the fluorographic detection of proteases after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The synthesis involved acetylation of Nϵ-benzyloxycarbonyl-L-lysine chloromethyl ketone with [3H]acetic anhydride just before the removal of the benzyloxycarbonyl group. By this method, [3H]ALCK with a specific activity of 250 mCi/mmol was obtained as a crystal. Trypsin, thrombin, plasmin, papain, and clostripain were inactivated by ALCK according to first-order kinetics. For fluorographic detection of proteases, enzyme samples were allowed to react with [3H]ALCK and then resolved by SDS-PAGE. Proteases that reacted with [3H]ALCK could be detected with a sensitivity equivalent to or higher than that of Coomassie brilliant blue R-250 staining. A trypsin-like protease in Pronase, clostripain as a contaminant in a commercial preparation of Clostridium histolyticzsm collagenase, and cysteine proteases in Porphyromonas gingivalis could be detected.

Using coomassie blue to stabilize H2O2-guaiacol stained peroxidases on polyacrylamide gels.

Shimoni and Reuveni [1] have reported a procedure which uses Coomassie Brilliant Blue R-250 staining and destaining to stabilize H2O2-guaiacol stained peroxidases on polyacrylamide gels and at the same time detects nonspecific protein bands. The procedure has the advantage that the otherwise orange and unstable peroxidase bands are visualized as blue bands and it, therefore, facilitates laser densitometry of electrophoretically separated peroxidases. In the modification of the procedure reported here, the Coomassie staining reagent was adjusted and the destaining step was eliminated. The modified procedure has added advantages: it simplifies the procedure, increases the consistency of the results across gels, prevents or reduces the staining of nonspecific proteins, and still gives excellent resolution of stable bands which can be used to quantify peroxidase activity.

Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis of human parotid salivary proteins

The proteins in human parotid saliva have been separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis into 20 or more well resolved species. The Coomassie Brilliant Blue (CBB) R-250 and silver staining procedures have been modified to overcome the problems encountered with staining of proline-rich proteins. By means of the CBB R-250 procedure which stains proline-rich proteins pink-violet, immunoblotting, concanavalin A binding, periodate-Schiff staining and zinc binding, all of the major proteins have been characterised. Substantial individual-to-individual differences were observed in the protein patterns formed. Comparison of parotid, submandibular, and whole saliva from a single individual indicated that fewer proline-rich proteins are expressed in submandibular saliva than in parotid, but whole saliva contains much lower levels than either duct secretion. The results will form a useful base for future research into the functions of salivary proteins.

Preparation by two-dimensional electrophoresis of proteins for antibody production: antibodies against proteins whose synthesis is reduced by auxin in tobacco mesophyll protoplasts.

Using phenol extraction from tobacco callus, we have prepared extracts with a high protein content. These proteins were separated in cylindrical non-equilibrium pH gradient gels and visualized by dipping in sodium dodecyl sulfate (SDS)-containing solution. Three gel sections, each containing proteins previously detected as abundantly synthesized in tobacco mesophyll protoplasts and whose synthesis is reduced by auxin application, were excised from each gel and collected. These proteins were further separated on slab SDS gels and protein bands were excised after Coomassie Brilliant Blue R-250 staining and used to inject three rabbits. After one booster, highly specific antibodies were detected in their sera by ELISA and immunoblotting. Using these sera we have confirmed that the corresponding proteins are identical in callus and mesophyll protoplast and demonstrated that they are abundantly accumulated in tobacco roots but are undetectable in aerial organs and seeds.

Staining of serum concanavalin a-binding proteins after micro two-dimensional electrophoresis and blotting

Human serum proteins separated by micro two-dimensional electrophoresis in the absence of denaturants were electrophoretically transferred to nitrocellulose sheets (blots), and glycoproteins on the blots were stained by sequential incubation with the lectin concanavalin A, the glycoprotein horseradish peroxidase and the substrates of peroxidase diaminobenzidine-hydrogen peroxide. The stained serum concanavalin A-binding glycoproteins were identified by referring to the “identification map” of human plasma proteins, which we have prepared by immunochemical staining of the proteins on the blots. The concanavalin A-peroxidase method applied for the blots after two-dimensional electrophoresis provided information on the location of serum concanavalin A-binding glycoproteins, and showed greater sensitivity than Coomassie Brilliant blue R-250 staining for several serum proteins.

An improved staining technique for precipitin bands in agar or agarose gels

A method for the staining of proteins in agar and agarose gels using three stains simultaneously and a mordant is described. When compared with conventional Coomassie brilliant blue R-250 staining procedures, it requires a comparable time expenditure but has the following advantages: 1) it is threefold to fourfold more sensitive; 2) there is increased photographic resolution on conventional photographic material; and 3) the stain has a long shelf-life and does not fade under normal lighting conditions. Conditions for the washing and drying of gels are discussed.

Antigenicity of the surface proteins of non o1 non o139 vibrio cholerae in rabbit model

An investigation was carried out to demonstrate the antigenicity of the surface proteins of the non O1 non O139 V. cholerae in rabbit model. Three rabbits were immunized with the surface proteins and sera were collected at one week intervals for six weeks. Sera were examined for antibodies specific for surface antigens using ELISA and the results showed a significant increase in antibody titers with time. The SDS?PAGE analysis showed a number of protein bands ranging from 21 to 131 kDa after Coomassie brilliant blue R250 staining. However, immunoblot analysis showed four major antigenic bands of 38, 43, 74 and 85 kDa to be prominent. These identified proteins might have higher immunogenic effects among humans infected with non O1 non O139 V. cholerae and some of these antigenic proteins could be included as diagnostic tools based on serology and also in vaccine preparations. Key words: Antigenicity; Surface proteins; Non O1 non O139 V. cholerae; Rabbit model DOI: http://dx.doi.org/10.3329/dujbs.v19i2.8954 DUJBS 2010; 19(2): 129-135