General Protein Staining Research Articles

Protein Stains and Applications.

Staining of proteins separated on gels provides the basis for determination of the critical properties of these biopolymers, such as their molecular weight and/or charge. Detection of proteins on gels and blots require stains. These stains vary in sensitivity, ease of use, color, stability, versatility, and specificity. This review discusses different stains and applications with details on how to use the stains, and advantages and disadvantages of each stain. It also compiles some important points to be considered in imaging and evaluation. Commonly used colorimetric and fluorescent dyes for general protein staining, and stains that detect posttranslational modification-specific detection methods are also discussed.

Detection of Proteins on Blot Membranes.

Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. © 2016 by John Wiley & Sons, Inc.

Analysis of oxidative stress-induced protein carbonylation using fluorescent hydrazides

Protein carbonyl detection has been commonly used to analyze the degree of damage to proteins under oxidative stress conditions. Most laboratories rely on derivatization of carbonyl groups with dinitrophenylhydrazine followed by Western blot analysis using antibodies against the dinitrophenyl moiety. This paper describes a protein carbonyl detection method based on fluorescent Bodipy, Cy3 and Cy5 hydrazides. Using this approach, Western blot and immunodetection are no longer needed, shortening the procedure and increasing accuracy. Combination of Cy3 and Cy5 hydrazides allows multiplexing analyses in a single two-dimensional gel. Derivatization with Bodipy hydrazide allows easy matching of the spots of interest and those obtained by general fluorescent protein staining methods, which facilitates excising target proteins from the gels and identifying them. This method is effective for detecting protein carbonylation in samples of proteins submitted to metal-catalyzed oxidation "in vitro" and assessing the effect of hydrogen peroxide and chronological aging on protein oxidative damage in yeast cells.

Stability of reference proteins in human placenta: General protein stains are the benchmark

The stability of reference proteins in semi-quantitative Western blot experiments in normal and diseased placenta has never been studied. This study aims to determine the stability of five reference proteins and two general protein stains in placentas from preeclampsia, gestational diabetes mellitus and matched control pregnancies. The stability of the reference proteins was analysed using indicators of inter-group (P value) and intra-group (coefficient of variation) stability. The effect of different normalization strategies was determined by normalizing serotonin transporter (SERT) expression against the different reference protein markers. Results show significant expression variability of β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1), peptidylprolyl isomerase A (PPIA) and α-tubulin, and that amido black staining is the most stable reference protein marker. Furthermore, results show that SERT expression significantly differs according to the reference protein markers used for its normalization. The present study demonstrated the importance of using stable reference protein markers and normalization strategy in order to get correct results in semi-quantitative Western blot experiments in placental tissues.

Post mortem development of meat quality as related to changes in cytoskeletal proteins of chicken muscles

1. A procedure was developed to separate high and medium molecular weight myofibrillar proteins from chicken muscular tissue with a high resolution by flat bed sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent detection by either a general protein stain or Western blotting. These procedures were used to analyse the degradation process of cytoskeletal proteins in chicken breast and leg muscles during meat ageing. 2. This study demonstrates the degradation of all the examined cytoskeletal proteins: titin, nebulin and desmin as well as vinculin, a protein component of the costamere structure. All the examined proteins were found to be degraded during ageing of chicken breast and leg muscles. 3. Degradation of titin, nebulin and desmin started at 3 h post mortem in breast muscle. Intact titin and nebulin disappeared within 1 d. Intact desmin and vinculin were not detectable after 3 d post mortem. In leg muscle, the degradation process of all the examined proteins evolved much more slowly than in breast chicken muscles. 4. The changes observed in shear force, myofibrillar fragmentation and cooking loss were related to changes in cytoskeletal proteins and used to identify marker proteins or degradation products for the purpose of monitoring the development of meat ageing. The ageing process was faster in breast muscle than in leg muscle. 5. Significant correlations were found between degradation processes of titin, nebulin, and desmin and shear force, as well as myofibril fragmentation index of breast and leg muscles.

Staining Proteins in Gels

This unit describes protocols for detecting protein in a gel by Coomassie blue, silver, or fluorescent staining. As a general protein stain, Coomassie is easier and more rapid; however, fluorescent and silver staining methods are considerably more sensitive and thus can be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and often as sensitive as silver staining. Alternate protocols describe rapid Coomassie and silver staining methods, as well as fluorescent stains that are specific for phosphoproteins and glycoproteins. Staining of proteins in SDS-polyacrylamide gels is described; variations for fluorescent staining of proteins in nondenaturing gels are also included. Support protocols describe photography of stained proteins.

Three new plasma protein polymorphisms in domestic foxes, detected by a simple method of 2D horizontal electrophoresis

Plasma samples of 270 foxes from 45 complete families (14 of arctic foxes, 28 of silver foxes and 3 with arctic x silver fox hybrid offspring) were analysed by a method of two-dimensional agarose gel (pH 5.4)-horizontal polyacrylamide gel (pH 9.0) electrophoresis followed by general-protein staining of gels. Genetic polymorphism of three plasma proteins, tentatively designated prealbumin (Pr), postalbumin 1 (Pa1), and pretransferin 1 (Prt1), was observed. In silver foxes, Pa1 and Prt1 showed a high degree of polymorphism, each with 3 common alleles, while Pr showed a scarce polymorphism. The arctic foxes were monomorphic for Pr and Prt1 and showed a scarce Pa1 polymorphism. The Prt1 phenotype of arctic foxes showed identical mobility with one of the Prt1 variants of silver foxes.

Differential protein labeling with thiol‐reactive infrared DY‐680 and DY‐780 maleimides and analysis by two‐dimensional gel electrophoresis

Differential protein labeling with 2-DE separation is an effective method for distinguishing differences in the protein composition of two or more protein samples. Here, we report on a sensitive infrared-based labeling procedure, adding a novel tool to the many labeling possibilities. Defined amounts of newborn and adult mouse brain proteins and tubulin were exposed to maleimide-conjugated infrared dyes DY-680 and DY-780 followed by 1- and 2-DE. The procedure allows amounts of less than 5 microg of cysteine-labeled protein mixtures to be detected (together with unlabeled proteins) in a single 2-DE step with an LOD of individual proteins in the femtogram range; however, co-migration of unlabeled proteins and subsequent general protein stains are necessary for a precise comparison. Nevertheless, the most abundant thiol-labeled proteins, such as tubulin, were identified by MS, with cysteine-containing peptides influencing the accuracy of the identification score. Unfortunately, some infrared-labeled proteins were no longer detectable by Western blots. In conclusion, differential thiol labeling with infrared dyes provides an additional tool for detection of low-abundant cysteine-containing proteins and for rapid identification of differences in the protein composition of two sets of protein samples.

Protein stains for proteomic applications:Which, when, why?

This review recollects literature data on sensitivity and dynamic range for the most commonly used colorimetric and fluorescent dyes for general protein staining, and summarizes procedures for the most common PTM-specific detection methods. It also compiles some important points to be considered in imaging and evaluation. In addition to theoretical considerations, examples are provided to illustrate differential staining of specific proteins with different detection methods. This includes a large body of original data on the comparative evaluation of several pre- and post-electrophoresis stains used in parallel on a single specimen, horse serum run in 2-DE (IPG-DALT). A number of proteins/protein spots are found to be over- or under-revealed with some of the staining procedures.

Rapid and easy protein staining for SDS-PAGE using an intramolecular charge transfer-based fluorescent reagent

High-performance staining for 1-D and 2-D SDS-PAGE was carried out using a novel protein-binding fluorophore (Dye 1), which noncovalently interacts with proteins and provides a fluorescence emission response to proteins by intramolecular charge transfer. In order to achieve the high-throughput analysis of proteins for SDS-PAGE, the general protocols for in-gel protein staining (SDS-PAGE, fixation, staining, washing, and detection) were simplified to produce an easy and rapid protocol (SDS-PAGE together with staining, washing, and detection). This method was performed by preparation of an electrophoresis buffer containing Dye 1 under optimum conditions, and by the binding of Dye 1 to proteins in the gel during the SDS-PAGE. As a result, this study required only 15 min for protein staining as a minimum time. On the other hand, it takes several hours for the general protein staining method, such as SYPRO Ruby staining (18 h) and CBB staining (105 min). Moreover, the protein-to-protein variation was low, and the detection limit was 7.0 ng/band of BSA (S/N = 3.0) in this method, which was as sensitive as the short-protocol silver staining methods. On the basis of these results, this rapid and easy protocol for SDS-PAGE using Dye 1 may be widely applicable and convenient for users in the various scientific and medical fields.

Functional characterization of two-dimensional gel-separated proteins using sequential staining

Proteins separated by two-dimensional (2-D) gel electrophoresis can be visualized using various protein staining methods. This is followed by downstream procedures, such as image analysis, gel spot cutting, protein digestion, and mass spectrometry (MS), to characterize protein expression profiles within cells, tissues, organisms, or body fluids. Characterizing specific post-translational modifications on proteins using MS of peptide fragments is difficult and labor-intensive. Recently, specific staining methods have been developed and merged into the 2-D gel platform so that not only general protein patterns but also patterns of phosphorylated and glycosylated proteins can be obtained. We used the new Pro-Q Diamond phosphoprotein dye technology for the fluorescent detection of phosphoproteins directly in 2-D gels of mouse leukocyte proteins, and Pro-Q Emerald 488 glycoprotein dye to detect glycoproteins. These two fluorescent stains are compatible with general protein stains, such as SYPRO Ruby stain. We devised a sequential procedure using Pro-Q Diamond (phosphoprotein), followed by Pro-Q Emerald 488 (glycoprotein), followed by SYPRO Ruby stain (general protein stain), and finally silver stain for total protein profile. This multiple staining of the proteins in a single gel provided parallel determination of protein expression and preliminary characterization of post-translational modifications of proteins in individual spots on 2-D gels. Although this method does not provide the same degree of certainty as traditional MS methods of characterizing post-translational modifications, it is much simpler, faster, and does not require sophisticated equipment and expertise in MS.

Variation in protein patterns within and among polychaete sibling species of the genus Marenzelleria (Spionidae): a preliminary survey

Differences in protein patterns of the soluble protein fraction among the sibling species Marenzelleria viridis (formerly type I) and M. neglecta (formerly type II) were investigated under common environmental conditions using two-dimensional gel electrophoresis (2D-PAGE). Protein expression was determined using general protein staining with Coomassie-blue and compared with radioactive labeling of proteins. In the well-resolved region of stained gels an average of 319 protein spots for M. viridis and 241 spots for M. neglecta could be detected. High sensitivity of radiolabeling allowed separation of an average of 517 and 496 spots for M. viridis and for M. neglecta, respectively. Differences in protein expression between both species could be attributed mainly to qualitative differences in protein patterns. Triplet pattern was used to calculate the genetic similarity of the two species. Thus, 373 protein spots were scored for this analysis; whereas 304 spots were invariant, 36 spots were specific for M. viridis, while 33 spots were specific for M. neglecta. The genetic similarity (F) of the two Marenzelleria sibling species was 0.815. Apart from presence and absence, differences between both species resulted either from slight changes in the isoelectric point or from molecular weight, but rarely from both. Genetic variability was found only among specimens of M. viridis. The experimental conditions to perform two-dimensional electrophoresis for these polychaete species were established for subsequent investigations on a proteomic level. Using 2D-PAGE we expect further insight into the evolutionary adaptation in Marenzelleria spp.

Selective proteome-wide detection of hydrophobic integral membrane proteins using a novel fluorescence-based staining technology.

Integral proteins containing two or more alpha-helical membrane-spanning domains are underrepresented in two-dimensional gels. While sodium dodecyl sulfate (SDS)-polyacrylamide gels separate these proteins, staining profiles are usually dominated by high-abundance hydrophilic proteins in the specimen. A fluorescence-based stain is presented that selectively highlights integral proteins containing two or more alpha-helical transmembrane domains but does not detect lipoproteins or proteins with hydrophobic pockets, such as albumin. The stain detects as little as 5-10 ng of bacteriorhodopsin, a seven-helix transmembrane protein. Stained proteins are detected using a laser scanner or charge-coupled device (CCD) camera imaging system. Fluorescence intensity of stained bands is linear with protein quantity over at least two orders of magnitude. After visualizing transmembraneous proteins, the total protein profile may be revealed using a general protein stain. Analysis of the multisubunit protein F1F0 ATP synthase revealed selective staining of the a and c subunits, polypeptides known to possess 5 and 2 transmembrane domains, respectively.

The unexpected outcomes of medical research: serendipity and the Australia antigenBlumberg BS, Alter HJ, Visnich S. A new antigen in leukemia sera [J Am Med Assoc 1965;191:541–546

The 'Australia Antigen' is found in the sera of some normal individuals from foreign populations. The total absence of the antigen from the sera of normal United States' subjects and its relatively high frequency in acute leukemia suggests that the presence of the antigen maybe of value in the diagnosis of early acute leukemia. Whether the antigen results from or precedes the leukemia process remains to be seen. (Abstract reproduced by permission of J Am Med Assoc 1965;191:541 - 546.) Many of the major discoveries in science have been the product of chance observations that were pursued by the curious mind to their plausible and provable explanation. Mendel's peas and, at least apocryphally, Newton's apple, come to mind as paradigm shifting observations that respectively opened the fields of genetics and established the laws of gravitation. Fleming's chance observation of the inhibition of bacterial growth in the presence of bread mold ultimately produced the first antibiotic. The unexpected link between the Australia antigen and the hepatitis B virus may be less encompassing than genetics or gravitation, but nonetheless is a classic example of a serendipitous observation pursued to major unexpected payoffs, including the prevention of post-transfusion hepatitis, the development of a vaccine, the prevention of hepatocellular carcinoma and laying the foundation for

Detection of Proteins on Blot Membranes

Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. © 2016 by John Wiley & Sons, Inc. Keywords: electroblots; electrotransfer efficiency; membrane stain; protein stain

A simple, rapid method for demonstrating bacterial flagella.

We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well as flagella and other appendages, which could be directly observed by epifluorescence microscopy. Detection of flagella was further improved by using a charge-coupled device camera for image capture and processing. The reliability of the method was tested by using 37 pure cultures of marine bacteria. Detection of flagella on the isolates by NanoOrange staining was compared to detection by transmission electron microscopy (TEM). For 36 of 37 cultures, the two methods yielded the same results. In one case, flagella were detected by TEM but not by NanoOrange, although the difference may be attributable to differences between the culture preparations. NanoOrange staining is rapid (10 to 15 min) and does not require fixation or dehydration, so live samples can be stained. Since NanoOrange is a general protein stain and works directly in seawater, it may also prove to be useful for staining other proteinaceous material that is of interest to aquatic microbial ecologists.

Diffusion blotting for rapid production of multiple identical imprints from sodium dodecyl sulfate polyacrylamide gel electrophoresis on a solid support

A simple and fast method for diffusion blotting of proteins from precast SDS-PAGE gels (0.5 mm) was developed. The efficiency of protein transfer was evaluated using 14 C labelled proteins. Diffusion blotting for three minutes, gave a transfer of 10% compared to electroblotting. By doubling the transfer time for each subsequent imprint, four imprints were made from the same lane with similar amounts of protein transferred onto each imprint. With a transfer time of three minutes each, it was possible to obtain at least ten imprints with all the proteins visible in all the imprints. There was no detectable loss in resolution as compared to electroblotting. The method also works well with an immuno-detecting system. The number of imprints which can be obtained, is dependent on the sensitivity of the detection system and the amount of protein applied. The greatest advantage of diffusion blotting compared to electroblotting is that several imprints can be made from each lane, and different antisera can be tested on identical imprints. The gel remains on its plastic support which prevents it from stretching and compression; this ensures identical imprints and facilitates more reliable molecular mass determination. If only a few imprints are made, sufficient protein remains within the gel for general protein staining. These advantages make diffusion blotting the method of choice when quantitative protein transfer is not required.

Soybean Protein Products as Regulators of Liver Low-Density Lipoprotein Receptors. I. Identification of Active β-Conglycinin Subunits

The ability of the different soy globulins to upregulate low-density lipoprotein (LDL) uptake and metabolism in human hepatoma cells (Hep G2) was investigated, in an attempt to identify peptide components responsible for the upregulation of the LDL receptor. In parallel, the metabolism of soy globulins, added to the culture medium, was investigated by two-dimensional electrophoresis. After addition of soy globulins, there were no marked changes in the cell protein pattern, as evaluated by general protein staining. By immunodetection, intact 7S components were observed both free in the culture medium and bound to plasma membranes. Inside cells, α + α‘ subunits in their native forms were not detectable, whereas most of the β chain was found unchanged. Largely unmodified soybean proteins were detected in a lysate of 11S-treated cells. Incubation of Hep G2 cell with purified α + α‘ from 7S sharply increased uptake and degradation of 125I-LDL added to the culture medium, whereas the β chains were ineffective; ...

State of the art of large-scale genetic purity testing of hybrid vegetable seeds using isoelectric focusing at PetoSluis.

During the past ten years we have been engaged in developing and applying isoelectric focusing techniques to test the genetic quality of vegetable seeds. We started with isoelectric focusing using carrier ampholytes (IEF-CA), and continued research with isoelectric focusing in immobilized pH gradients (IEF-IPG) and two-dimensional electrophoresis (IPG-DALT). In addition, we have developed equipment and procedures for large-scale seed and seedling homogenization, sample preparation and semi-automatic gel staining. Moreover, we have optimized the sample application and gel running setup for large-scale analysis. We have developed hybrid purity (inbred) testing methods for all important vegetables, e.g., melon, cole crops, tomato, pepper, watermelon, squash, cucumber, radish etc. using either IEF-CA or IEF-IPG of seed or seedling proteins, followed by specific or general protein staining. To indicate the efficiency of the equipment and procedures developed we present results of two of our hybrid purity test methods, namely for brassica using polymorphism for phosphoglucomutase (PGM) from dry seeds, and for tomato using polymorphism for alcohol dehydrogenase (ADH) from imbibed seeds. We show that one person can routinely analyze 1536 individual seeds per day at a cost of about US +0.11 per seed for chemicals, materials, and electrophoresis equipment.

A sensitive method for detection of proteins in polyacrylamide gels and on protein blots using acidic henna leaf extract

In the present communication we describe a process for the preparation of an acidic henna leaf extract useful as a general protein stain for both polyacrylamide gels and protein blots. The staining is reversible by changing its pH and does not require protein fixation or destaining steps. This staining procedure is simpler and also, its sensitivity is comparable with the two commonly used organic stains i.e. Coomassie Blue and Amido Black. Under optimum conditions the protein detection limits of acidic henna leaf extract varied from 50 ng to 100 ng.