Comparison of the Coomassie brilliant blue, bicinchoninic acid and Lowry quantitation assays, using non-glycosylated and glycosylated proteins.

The concentrations of several non-glycosylated and glycosylated recombinant and native proteins were determined by three widely used colorimetric methods: Coomassie brilliant blue, bicinchoninic acid and Lowry, and, for comparison, by amino acid composition analysis. The colorimetric methods gave results differing from the values derived from the amino acid analysis, in some cases by up to 60%. For the non-glycosylated recombinant proteins, the results were in relatively good agreement with each other and with the values determined on the basis of the amino acid analysis. The Coomassie blue method was strongly dependent on the hydrophobicity of the individual protein. The bicinchoninic acid method gave results closest to those of the amino acid analysis. For the glycosylated proteins, both recombinant and native, the Coomassie blue assay gave values lower, whereas the two other methods gave values higher than those determined on the basis of the amino acid analysis. The concentration of a recombinant interferon gamma receptor produced in two differently glycosylated forms was underestimated by the Coomassie blue assay and overestimated by the bicinchoninic acid and Lowry methods, while for the non-glycosylated form of the same protein, the three colorimetric methods delivered comparable values. The results suggest a potential interference of protein glycosylation with the colorimetric assays.     

Semipreparative electrophoresis with prestained proteins.

A simple method for prestaining proteins with Coomassie blue G prior to acrylamidegel electrophoresis is described. Many, although not all, undenatured proteins can be stained. This prestaining technique can be conveniently coupled with a second electrophoresis step, using Sephadex as a supporting medium, to yield isolated protein in about 4 h.     

Decreased Coomassie brilliant blue colour yield with glycated proteins.

Collagen, myosin and albumin were incubated for 7 days at 20 degrees C with fructose, ribose or glyceraldehyde. For thus-formed glycated proteins, quantities were determined by the Conway microdiffusion technique and by the colorimetric method based on Coomassie brilliant blue G-250 colour yield. It was found that when albumin was glycated with increasing amounts of glyceraldehyde, the colour yield was decreased by 7-33%. In collagen, myosin and albumin incubated with 0.5 mol/l fructose, 0.5 mol/l ribose or 0.1 mol/l glyceraldehyde, protein concentration was not changed, as proved by the Conway microdiffusion technique; the Coomassie brilliant blue G-250 colour yield was up to 50% lower, depending on the protein used, and was decreased much less when proteins were incubated with less sugar.     

Identification of the milk fat globule membrane proteins. II. Isolation of major proteins from electrophoretic gels and comparison of their amino acid compositions

The fat globule membranes of milk are derived from the apical plasma membrane of the mammary secretory cells. The nature of the membrane proteins, as isolated from cows' milk, has been studied by the use of discontinuous and continuous SDS-gel electrophoresis. Six methods of preparation of milk fat globule membrane suggested by various authors were tested; gel electrophoresis showed that five major bands were present, independent of the method of preparation. The apparent molecular masses of these proteins as determined on SDS-gels (15% T) were 167, 142, 64, 49 and 46 kDa, respectively. The 167 kDa band stained only with periodic acid-Schiff reagent, while the 142 kDa band stained only with Coomassie blue; the last three bands stained with both. Delipidated membranes were extracted stepwise with water, 0.02 M NaCl and 0.6 M NaCl. The 64 kDa band appears to be nearly insoluble, while the bands of 142, 49 and 46 kDa are fractionated by this procedure. The resolution of all of these proteins by electrophoresis was superior to that achieved by molecular sieve chromatography, and so electrophoretic extraction was used to isolate the major proteins. Dansyl chloride derived proteins were used as markers. Amino acid compositions of the recovered proteins were obtained and are compared.     

Detection by the use of silver ions of additional protein zones in gradient polyacrylamide gels stained with Coomassie

A simple method of revealing the additional zones of proteins in gradient polyacrylamide gels, preliminary dyed Coomassie by means of silver ions is described. The dyeing of Coomassie allows to avoid the time-consuming stages of preliminary treatment of gels as well to reveal more sensitive zones in gels. On the second stage of dyeing silver minor zones appear there which were not seen while Coomassie was dyed. The suggested method preserves high sensitivity characteristic of the methods of gel dyeing with silver.     

Ribosomal proteins from rabbit reticulocytes: number and molecular weights of proteins from ribosomal subunits.

The ribosomal proteins from 40 S and 60 S subunits of rabbit reticulocytes were separated by two-dimensional polyacrylamide gel electrophoresis. The protein spots stained with Coomassie brilliant blue were cut out and the proteins were extracted. The material extracted from each spot was mixed with proteins of known molecular weight and then analyzed by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Both the total number and the molecular weights of each of the proteins were determined by these procedures. Thirty-two proteins were identified in the 40 S subunits; their molecular weights ranged from 8000 to 39,000 (average mol. wt = 25,000). Thirty-nine proteins were identified in the 60 S subunit; their molecular weights ranged from 9000 to 58,000 (average mol. wt = 31,000). The sum of the molecular weights of the individual proteins from each subunit is in agreement with previous estimations, derived from physico-chemical measurements of the total protein in mammalian ribosomal subunits. The molecular weight distribution obtained for the isolated proteins was nearly identical to that derived from spectrophotometric analysis of polyacrylamide-sodium dodecyl sulfate gels of the total protein mixtures from each subunit stained with Coomassie brilliant blue. The results are consistent with the hypothesis that reticulocyte ribosomes contain one copy of most of their protein constituents.     

Biliary proteins: assessment of quantitative techniques and comparison in gallstone and nongallstone subjects

Although protein is the third most abundant solid in bile and is important in cholesterol crystal formation, methods for quantitating the concentration of total protein in bile have not been systematically evaluated. To establish a reliable protein assay for bile, we evaluated three protein assays (Lowry's method and the fluorescamine and Coomassie blue methods), and employed amino acid analysis as a reference technique. Large protein-to-protein variations were observed with the fluorescamine and Coomassie blue methods. Although all assays were affected by interfering substances, Lowry's method and the fluorescamine technique (after trichloroacetic acid precipitation and delipidation of bile) and the Coomassie blue method with native bile showed excellent correlations (P less than 0.0001) with those obtained by amino acid analysis. Using these reliable protein assays, we examined gallbladder bile obtained at surgery from subjects with and without gallstones. No differences in the concentrations of total biliary proteins were observed among patients with cholesterol (n = 23) or pigment (n = 7) gallstones and subjects without gallstones (n = 10). Protein values obtained by amino acid analysis also did not differ among groups. As expected, bile from patients with cholesterol gallstones was supersaturated with cholesterol while bile from nongallstone subjects and those with pigment stones was unsaturated. These results indicate that it is not possible to separate patients with and without gallstones on the basis of the total protein concentration of gallbladder bile.     

Identification and characterization of sex-linked proteins of Schistosoma mansoni.

The proteins of adults worms (male and female) of two isolates (BH and RJ) of Schistosoma mansoni were extracted using Triton X-114 phase separation. The SDS-polyacrilamide gel electrophoresis profiles of the three phases (detergent, aqueous and insoluble proteins) obtained were compared after Coomassie blue and silver staining, surface radioiodination and Western blotting. No major differences were detected between the 2 isolates. Of the 25 or more proteins which partitioned into the detergent phase, only about 8 proteins could be surface radiodinated on live adult worms. A comparison was also made between the profiles of male and females worms, isolated from bisexually infected mice. Two major female-specific and one male-specific band were detected by silver and/or Coomassie staining. The female bands, 32 KDa and 18 KDa, partitioned into the detergent and aqueous phase, respectively. The male-specific band of 42 KDa remained in the insoluble phase. Antigenic differences between male and females proteins were detected by Western blotting using a sera from infected Nectomys squamipes.     

A faithful double stain of proteins in the polyacrylamide gels with Coomassie blue and silver

The conditions for prior fixing of proteins in a gel in order to attain a greater degree of faithful silver staining and sensitivity were examined. Fixing with formaldehyde enhanced the retention of proteins in a gel, particularly basic proteins such as histones and ribosomal proteins. The gel, one stained with Coomassie blue and following the removal of the free dyes, is capable of undergoing silver staining, and, moreover, the prestain considerably enhanced the staining intensity of various proteins differing in basicity in subsequent silver staining. Coupling the formaldehyde fixation with Coomassie brilliant blue prestain afforded a reproducible and pronounced stainability of various proteins.     

Quantitation of stained proteins in SDS polyacrylamide gels with lysozyme as internal standard.

A simple method for the quantitation of proteins on SDS-polyacrylamide gels, with lysozyme as an internal standard, has been designed. Gels containing known weight ratios of standard proteins to lysozyme were electrophoresed, stained with Coomassie blue R250, and scanned at 550 nm. Peak areas corresponding to individual proteins were determined and the area ratios of proteins to lysozyme were calculated. Plots of area ratio vs weight ratio were linear over a limited range and were reproducible from gel to gel and thus suffice as a standard curve. We have used this method to determine accurately and precisely the amount of rhodopsin in the photoreceptor membranes of rat retinas.     

Selective silver-staining methods for RNA and proteins in the same polyacrylamide gels

Two silver-staining methods for selective and ultrasensitive detection of RNAs and proteins in the same polyacrylamide gels were developed, both derived from procedures recommended for protein staining. The first, a double-staining technic with Coomassie brilliant blue and ammoniacal silver, allows visualization of RNAs as negative bands and proteins as dark brown bands. The second is also a double-staining technique, but uses silver in both steps. This second method develops the RNA bands first and then the protein bands. These techniques, especially the second, permit characterization of the different components of ribonucleoproteic complexes in the same electrophoresis gels.     

Production and separation of peptides from proteins stained with Coomassie brilliant blue R-250 after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Proteins stained with Coomassie brilliant blue on polyacrylamide gels were digested with lysylendopeptidase in the presence of sodium dodecyl sulfate. Peptide production was similar to that under ordinary conditions of digestion. Peptides were recovered easily and efficiently from the gel pieces and separated by HPLC. The present method for preparation of peptides from proteins separated by sodium dodecyl sulfate gel electrophoresis is quite simple and can be used for sequence analysis of proteins in general at the subnanomolar level.     

Staining of free sulfhydryl groups of proteins after separation by isoelectric focusing of Bio-Rad standards and lens crystallins.

In this study, we propose a new staining method for free sulfhydryl groups of proteins after having separated native samples by thin-layer isoelectric focusing (IEF) in absence of detergents. A comparison was made between proteins stained purple for free sulfhydryl groups (SH) and proteins stained blue by Coomassie blue (CB). A stainability factor, F = %SH/%CB, was calculated for each protein. The Bio-Rad IEF standards containing seven marker proteins for pH scale determination were stained purple, in the same way as they were designed for CB staining. To prove the validity of the currently proposed staining method for a defined protein system such as the eye lens crystallins, these proteins were also stained after IEF as described above. The factor F was calculated for all alpha-, beta-, and gamma-crystallin components that stained in both methods. We discovered that alpha-crystallins contained comparatively high amounts of free SH groups, while some beta- and gamma-crystallin components also contained considerable amounts of free SH groups. The SH staining procedure with 2,2'-dihydroxy-6,6'-dinaphthyl disulfide applied after IEF appeared to be useful, specific, and reproducible.     

Characterization of the cilia and ciliary membrane proteins of wild- type Paramecium tetraurelia and a pawn mutant

Cilia and ciliary membranes were isolated from axenically grown, wild- type Paramecium tetraurelia strain 51s and from the extreme pawn mutant strain, d495, derived from this parental strain. Over 60 protein bands having molecular weights of 15 to greater than 300 kdaltons were detected by Coomassie Blue staining of whole cilia proteins separated by one-dimensional SDS polyacrylamide gel electrophoresis. About 30 of these protein bands were visible in Coomassie Blue-stained membrane separations. About 60 bands were detected by silver staining of one- dimensional gels of membrane proteins. Differences between Coomassie Blue-stained separations of wild-type and pawn mutant strain d495 membrane proteins were seen in the quantity of a band present at 43 kdaltons. Radioiodination of cell surface proteins labeled approximately 15 protein bands in both wild-type and mutant cilia. The major axonemal proteins were unlabeled. Six membrane glycoproteins were identified by staining one-dimensional separations with iodinated concanavalin A and lentil lectin, two lectins that specifically bind both glucose and mannose residues. Two major neutral sugar species present in an acid hydrolysate of the cilia preparation were tentatively identified as glucose and mannose by gas chromatography of the alditol acetate derivatives.     

Imidazole-SDS-Zn reverse staining of proteins in gels containing or not SDS and microsequence of individual unmodified electroblotted proteins.

A reverse staining procedure is described for the detection of proteins in acrylamide and agarose gels with and without SDS. Protein detection occurs a few minutes after electrophoresis. The sensitivity on acrylamide gels is higher than that of Coomassie blue staining either on acrylamide gels or on electrotransferred membranes. Sequencing of protein bands only detected by reverse staining on the gel and not by Coomassie blue is demonstrated.     

Alterations in surface proteins during myogenesis of a rat myoblast cell line.

Lactoperoxidase-catalyzed iodination and SDS-polyacrylamide gradient gel electrophoresis were used to examine the surface proteins of cultures of an embryonic rat myoblast cell line during myogenesis. We observed several consistent alterations in the proteins iodinated during the periods of alignment and fusion. In addition, we examined the surface proteins of cultures where fusion was inhibited by phospholipase C (PLC), and of cultures of several nonfusing variants of our original line. Many of the proteins which appeared during “normal” myogenesis were not seen in PLC-treated cultures, while the appearance and loss of three low molecular weight proteins were accelerated. The nonfusing variants often accumulated large amounts of many of the proteins which appeared during alignment in normal cultures. This accumulation was demonstrated by Coomassie blue stain intensities as well as by the extent of surface iodination. The three low molecular weight proteins were heavily iodinated in one class of variant, but did not disappear as in normal cultures. One protein of apparent molecular weight 66,000 (66K) was iodinated during alignment but was inaccessible during fusion. Coomassie blue staining of the gels showed that the actual appearance and disappearance of the 66K protein band from the membrane were coincident with alignment and fusion. While this band was not seen in fluorograms from gels of PLC-treated cultures and some of the nonfusing variants, a 66K band was invariably stained by Coomassie blue, and in PLC-treated cultures appeared to accumulate with time. In the variants there appeared to be a correlation between the availability of the 66K protein for iodination and the appearance of the low molecular weight proteins.     

Eosin Y staining of proteins in polyacrylamide gels.

A staining method is described in which various proteins in polyacrylamide gels can be stained by using eosin Y. After a brief incubation of a polyacrylamide gel in an acidic solution of 1% eosin Y, various proteins, including human erythrocyte membrane sialoglycoproteins which are not detectable by Coomassie blue R-250 (CB), can be detected with a sensitivity of 10 ng protein. This is far more sensitive than CB staining and is comparable to the sensitivity of silver staining. In a Western blot, the antigenicity of an eosin Y stained protein is retained. In addition, proteins on an immunoblot sheet can be detected by eosin Y staining. The method described is rapid, sensitive, and reproducible with various proteins in polyacrylamide gels and has the added advantage of also staining sialoglycoproteins.     

A rapid and reproducible assay for quantitative estimation of proteins using bromophenol blue

A method for the quantitation of proteins in solution which involves the binding of bromophenol blue to proteins under acidic conditions has been developed. The binding of the dye to proteins is accompanied by the appearance of a strong absorbance at 610 nm, which is almost linear over the range of 10 to 80 μg for the seven proteins studied. The absorbance at 610 nm can be measured immediately after the mixing of the protein and dye solutions and is stable over a period of 8 hr. The method has very few interferences, most of which can be corrected for by the use of proper controls. Phenol, sodium dodecyl sulfate, and Triton X-100 (the last with some error) may be used with this assay at concentrations that produce strong interferences with similar methods using Coomassie brilliant blue G-250.     

Histone and nonhistone proteins from normal and virus-transformed rat thyroid epithelial cells

The histone and nonhistone nuclear proteins extracted by different methods from nuclei of normal and virus-transformed rat thyroid epithelial cells (FRTL5 cell line) were studied by polyacrylamide gel electrophoresis in acetic acid/urea and in SDS and by autoradiography. The results have shown that some of these proteins have an higher level of phosphorylation in virus-transformed than in normal cells; moreover, an higher amount of three proteins (C, D, and E), which in normal cells are not detectable at least as Coomassie staining, was found. These proteins, extractable with perchloric acid, are suggested to belong to the High Mobility Group (HMG proteins) and to play some regulatory role.