Microwave-assisted and methanol/acetic acid-free method for rapid staining of proteins in SDS-PAGE gels.

We describe a microwave-assisted, methanol and acetic acid-free, inexpensive method for rapid staining of SDS-PAGE proteins. Only citric acid, benzoic acid, and Coomassie brilliant blue G-250 (CBG) were used. Microwave irradiation reduced the detection duration, and proteins in a clear background were visualized within 30 min of destaining, after 2 min of fixing and 12 min of staining. By using this protocol, comparable band intensities were obtained to the conventional methanol/acetic acid method.

RGBradford: Accurate measurement of protein concentration using a smartphone camera and the blue to green intensity ratio.

The color shift caused by the interaction between proteins and Coomassie Brilliant Blue in the Bradford assay can be recorded with a smartphone camera. Color data can then be extracted from pictures and processed as analytical signals. Here, I present a simple procedure to accurately measure protein levels using color data from pictures of microplates. Plotting the ratio of blue to green intensity (RGB scale) as a function of protein concentration results in a linear (R2 ≥ 0.99) relationship, from which protein levels in biological samples can be calculated with no significant difference from values obtained using absorbance data (RGB versus absorbance curves).

Aptamer-modified magnetic SERS substrate for label-based determination of cardiac troponin I.

A sensitive label-based SERS strategy composed of magnetic bimetallic nanoparticles Fe3O4@Ag@Au, specific aptamer, and Bradford method was developed for the quantitative determination of cardiac troponin I (cTnI) in human serum. The prepared substrate with high magnetic character, signal enhancement, and uniformity exhibited significant Raman response. After the substrate was bound to the aptamer, the target protein cTnI was specifically captured, and it showed the Raman signal when the signal reporter Coomassie Brilliant Blue G-250 (CBBG) was supplied. The Raman signal intensity at 1621 cm-1 showed a wide linear relationship with the log value of the cTnI concentration in the range 0.01 to 100 ng·mL-1, and the estimated limit of detection (LOD) was 5.50 pg·mL-1. The recovery and relative standard deviation (RSD) of the spike experiment in human serum samples were 92-115% and 7.4-12.7%, respectively.

PEPPI-MS: Polyacrylamide-Gel-Based Prefractionation for Analysis of Intact Proteoforms and Protein Complexes by Mass Spectrometry.

Prefractionation of complex mixtures of proteins derived from biological samples is indispensable for proteome analysis via top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis (PAGE), which enables high-resolution protein separation based on molecular size, is a widely used technique in biochemical experiments and has the potential to be useful in sample fractionation for top-down MS analysis. However, the lack of a means to efficiently recover the separated proteins in-gel has always been a barrier to its use in sample prefractionation. In this study, we present a novel experimental workflow, called Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS ("PEPPI-MS"), which allows top-down MS of PAGE-separated proteins. The optimization of Coomassie brilliant blue staining followed by the passive extraction step in the PEPPI-MS workflow enabled the efficient recovery of proteins, separated on commercial precast gels, from a wide range of molecular weight regions in under 10 min. Two-dimensional separation combining offline PEPPI-MS with online reversed-phase liquid chromatographic separation resulted in identification of over 1000 proteoforms recovered from the target region of the gel (≤50 kDa). Given the widespread availability and relatively low cost of traditional sodium dodecyl sulfate (SDS)-PAGE equipment, the PEPPI-MS workflow will be a powerful prefractionation strategy for top-down proteomics.

Urine protein quantification in stacking gel by SDS-PAGE.

Urine total protein concentration is usually measured by the pyrogallol red-molybdate (PRM) assay in clinical laboratories, but it is often subject to sample interference. Here, we introduce a stacking gel-based method for accurate protein quantitation. In this method, the urine protein samples are run into the stacking gel by SDS-PAGE where it is concentrated into a single band, and then quickly stained by 0.001% Coomassie at high temperature. High correlations were found between the BSA and urine protein standards (R2 = 0.997 and 0.990, respectively). Addition of 80 ng urine protein standard into each of the ten clinical urine specimens with questionable PRM results yielded the expected increase in the results by this method. Comparison of the PRM method and with the gel quantitation approach on about 60 clinical urine samples demonstrated a general consistency between the results (R2 = 0.825), but in PRM samples with lower protein concentration showed more variations. Overall, the stacking gel method might be a good alternative for clinical urine samples with suspicious protein concentration results.

Quantitation of low concentrations of polysorbates 80 in protein formulations by Coomassie brilliant blue.

Polysorbate 80, as pharmaceutical excipient and virus inactivating agent, is commonly used in the protein pharmaceutical industry. In this study, a method has been developed for the determination of low concentration of Polysorbate 80 in the presence of high concentration proteins (≤100 mg/ml) and excipients. This colorimetric method is based on the interaction of Polysorbate 80 and Coomassie brilliant blue, and suitable for quantitation of Polysorbate 80 in the range of 10-100 µg/ml. Dozens or hundreds of samples can be quantified simultaneously by using microplate. Besides Polysorbate 80, this method can also be used to determine other types of surfactants in protein solutions, such as Polysorbate 20, Triton X-100, NP40, SDS, Benzalkonium chloride/bromide and PEG4000.

Crystal violet stains proteins in SDS-PAGE gels and zymograms.

Coomassie brilliant blue R250, an anionic dye is the most popular stain to detect proteins resolved in SDS-PAGE gels. Crystal violet, a cationic dye was found to be versatile and stained proteins in SDS-PAGE gels and in zymograms. Stained proteins can be transferred to nitrocellulose and the stained proteins on the western detected with enzyme coupled antibodies. Staining can be reversed. Staining takes 3 h at RT or 30 min at 60 °C. Crystal violet stained some E. coli high and low molecular weight proteins not stained by Coomassie blue R250. Crystal violet stained down to 16 ng of protein, some five-fold lower than Coomassie blue, though the two stains had a similar linear dynamic range. The staining sensitivity could be increased to 2 ng when crystal violet and Coomassie blue were combined in a double staining/counterion dye formulation. The low concentrations of the dye without a destaining step reduces the costs of the technique and results in a more environmentally friendly stain compared to traditional staining methods.

Green synthesis of CuO nanomaterials and their proficient use for organic waste removal and antimicrobial application.

Copper oxide (CuO) nanomaterials (NMs) of different size and morphology were synthesized by Chemical precipitation, Microwave irradiation and Hydrothermal method and characterized by TEM, BET, FTIR, XRD and EDX analysis. As synthesized CuO NMs were utilized for elimination of harmful dyes viz. Direct Red 81 (DR-81) and Coomassie Brilliant blue R-250 (BBR-250) and pathogenic bacteria (Staphylococcus aureus). Owing to their morphology, smaller size and relatively high surface area (40.320 m2 g-1), CuO NMs prepared by chemical precipitation method were observed to show better adsorption capacity for both the dyes (68.70 (DR-81) and 73.04 (BBR-250) mg g-1). The influence of different experimental conditions was studied by the methodical assessments of various parameters such as pH, adsorbent dose, concentration and contact time. Moreover, different adsorption isotherms and pseudo-second order kinetic model were applied to understand the adsorption mechanism. Langmuir model was found to be best fit thus confirming the monolayer adsorption process. To ensure the practical utility of CuO NMs for organic waste removal, the adsorption studies were performed in the presence of different inorganic ions and real water samples. In addition, recovery of the dye and NMs were also carried out effectively by simple method, thus avoiding the secondary pollution. CuO NMs were observed to exhibit significant antibacterial activity against the human pathogenic bacteria. These studies demonstrated that synthesized CuO NMs showed good adsorption efficiency for the removal of harmful dyes and antimicrobial activity against the pathogenic bacteria, which vary as a function of size and surface area.

Specific glycine to alanine mutation eliminates dynamic interaction of polymeric GlyT1a N-terminus with Coomassie Brilliant Blue G-250.

We previously found that multimeric GlyT1aN16 protein exhibits increased diffusion in a polyacrylamide gel and shows an unusual time-dependent absorbance rearrangement, as revealed by the Bradford assay. Here, we find that glycine to alanine mutation eliminates the absorbance shift, but not the altered diffusion properties of GlyT1aN16, indicating that these two phenomena are not interconnected. The absorbance shift is apparent with both native and urea-denatured GlyT1aN16, suggesting that the effect is either not dependent on protein structure, or the required structure is restored very quickly following denaturant removal. In the far-UV spectra, circular dichroism (CD) curves for both wild-type and mutated GlyT1aN16 are under the zero line, indicating largely unstructured character. However, a significant shift of the mutant CD curve suggests possible microstructural heterogeneity. Deconvolution of the CD data indicates a potential 3-fold increase in isolated helical content, which would inhibit an absorbance shift, as we demonstrated previously. These results suggest that, in addition to protein quantification, Coomassie Brilliant Blue G-250 can be used to reveal certain properties of the secondary structure of proteins.

Coomassie staining provides routine (sub)femtomole in-gel detection of intact proteoforms: Expanding opportunities for genuine Top-down Proteomics.

Modified colloidal Coomassie Brilliant Blue (cCBB) staining utilising a novel destain protocol and near-infrared fluorescence detection (nIRFD) rivals the in-gel protein detection sensitivity (DS) of SYPRO Ruby. However, established DS estimates are likely inaccurate in terms of 2DE-resolved proteoform 'spots' since DS is routinely measured from comparatively diffuse protein 'bands' following wide-well 1DE. Here, cCBB DS for 2DE-based proteomics was more accurately determined using narrow-well 1DE. As precise estimates of protein standard monomer concentrations are essential for accurate quantitation, coupling UV absorbance with gel-based purity assessments is described. Further, as cCBB is compatible with both nIRFD and densitometry, the impacts of imaging method (and image resolution) on DS were assessed. Narrow-well 1DE enabled more accurate quantitation of cCBB DS for 2DE, achieving (sub)femtomole DS with either nIRFD or densitometry. While densitometry offers comparative simplicity and affordability, nIRFD has the unique potential for enhanced DS with Deep Imaging. Higher-resolution nIRFD also improved analysis of a 2DE-resolved proteome, surpassing the DS of standard nIRFD and densitometry, with nIRFD Deep Imaging further maximising proteome coverage. cCBB DS for intact proteins rivals that of mass spectrometry (MS) for peptides in complex mixtures, reaffirming that 2DE-MS currently provides the most routine, broadly applicable, robust, and information-rich Top-down approach to Discovery Proteomics.

A new procedure for fast soft staining of BN-PAGEs on photosynthetic complexes.

We report a fast and sensitive procedure for blue native PAGE staining, in which the conventional staining step with CBB is avoided. After running, a short exposure to a mix of polar protic solvents (ethanol and acetic acid) leads to a fast and selective removal of the dye from the migration front and a specific binding to the protein bands, while the rest undergo a selective and complete background removal, leading to an intense contrast. This single-step staining-destaining technique is useful in protein samples that bind colored cofactors such as photosystems, which can be selectively discerned by their characteristic green color. After the staining of such samples, the green color persists, while the other unpigmented protein complexes and the molecular standard remain CBB stained, creating a useful reference system for the assignment of the bands. The advantages and chemical basis of this staining procedure are discussed.

In-situ protein determination to monitor contamination in a centrifugal partition chromatograph.

Centrifugal partition chromatography (CPC) works with biphasic liquid systems including aqueous two-phase systems. Metallic rotors are able to retain an aqueous stationary phase able to purify proteins. But the adhesion of proteins to solid surface may pose a cross-contamination risk during downstream processes. So it is of utmost importance to ensure the cleanliness of the equipment and detect possible protein contamination in a timely manner. Thereby, a direct method that allows the determination of the effective presence of proteins and the extent of contamination in the metallic CPC rotors was developed. This in-situ method is derived from the Amino Density Estimation by Colorimetric Assay (ADECA) which is based on the affinity of a dye, Coomassie Brillant Blue (CBB), with protonated N+ groups of the proteins. In this paper, the ADECA method was developed dynamically, on a 25 mL stainless-steel rotor with various extents of protein contaminations using bovine serum albumin (BSA) as a fouling model. The eluted CBB dye was quantified and found to respond linearly to BSA contamination up to 70 mg injected. Limits of detection and quantification were recorded as 0.9 mg and 3.1 mg, respectively. While the non-specific interactions between the dye and the rotor cannot currently be neglected, this method allows for in situ determination of proteins contamination and should contribute to the development of CPC as a separation tool in protein purification processes.

Exopolymer production as a function of cell permeability and death in a diatom (Thalassiosira weissflogii) and a cyanobacterium (Synechococcus elongatus).

Exopolymer particles are found throughout the ocean and play a significant biogeochemical role in carbon cycling. Transparent exopolymer particles (TEP) are composed of acid polysaccharides, and Coomassie staining particles (CSP) are proteins. TEPs have been extensively studied in the ocean, while CSP have been largely overlooked. The objective of this research was to determine the role of stress and cell permeability in the formation of TEP and CSP. The diatom Thalassiosira weissflogii and cyanobacterium Synechococcus elongatus were grown in batch cultures and exposed to hydrogen peroxide (0, 10, and 100 µM) as an environmental stressor. There was no correlation between TEP and CSP concentrations, indicating that they are different populations of particles rather than different chemical components of the same particles. CSP concentrations were not affected by hydrogen peroxide concentration and did not correlate with indicators of stress and cell death. In contrast, TEP concentrations in both taxa were correlated with a decrease in the effective quantum yield of photosystem II, increased activity of caspase-like enzymes, and an increase in the proportion of the population with permeable cell membranes, indicating that TEP production was associated with the process of cell death. These data show that different environmental factors and physiological processes affected the production of TEP and CSP by phytoplankton. TEP and CSP are separate populations of exopolymer particles with potentially different biogeochemical roles in the ocean.

Coomassie Blue G250 for Visualization of Active Bacteria from Lake Environment and Culture.

Bacteria play a fundamental role in the cycling of nutrients in aquatic environments. A precise distinction between active and inactive bacteria is crucial for the description of this process. We have evaluated the usefulness of Coomassie Blue G250 for fluorescent staining of protein containing potentially highly active bacteria. We found that the G250 solution has excitation and emission properties appropriate for direct epifluorescence microscopy observations. It enables fast and effective fluorescent visualization of living, protein-rich bacteria, both in freshwater environment and culture. Our results revealed that the number of G250-stained bacteria from eutrophic lake was positively correlated with other standard bacterial activity markers, like number of bacteria containing 16S rRNA, bacterial secondary production or maximal potential leucine-aminopeptidase activity. In case of the E. coli culture, the percentage of bacteria visualized with G250 was similar to that of bacteria which accumulated tetracycline. Compared to other common methods utilizing fluorogenic substances for bacteria staining, the approach we evaluated is inexpensive and less hazardous (for example mutagenic) to the environment and researchers. It can be regarded as an additional or alternative method for protein rich, active bacteria staining.

Destaining of Coomassie Brilliant Blue R-250-stained polyacrylamide gels with fungal laccase.

An enzyme-based method for destaining polyacrylamide gels stained with Coomassie Brilliant Blue R-250 is described. Distilled water supplemented with diluted fermentation broth of a laccase-producing white-rot fungus, Cerrena sp., was used for gel destaining, and a clear gel background was obtained in 2 h at 37 °C. Sensitivity of protein detection was 10 ng. The method did not require organic solvents or changing the destaining solution. Due to simultaneous gel destaining and dye decolorization, the colorless destaining solution can be disposed of directly. Laccase destaining of polyacrylamide gels was simple, efficient, and environmentally friendly.

Total protein or high-abundance protein: Which offers the best loading control for Western blotting?

Western blotting routinely involves a control for variability in the amount of protein across immunoblot lanes. Normalizing a target signal to one found for an abundantly expressed protein is widely regarded as a reliable loading control; however, this approach is being increasingly questioned. As a result, we compared blotting for two high-abundance proteins (actin and glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) and two total protein membrane staining methods (Ponceau and Coomassie Brilliant Blue) to determine the best control for loading variability. We found that Ponceau staining optimally balanced accuracy and precision, and we suggest that this approach be considered as an alternative to normalizing with a high-abundance protein.

Involvement of Val 315 located in the C-terminal region of thermolysin in its expression in Escherichia coli and its thermal stability.

Thermolysin is a thermophilic and halophilic zinc metalloproteinase that consists of ß-rich N-terminal (residues 1-157) and α-rich C-terminal (residues 158-316) domains. Expression of thermolysin variants truncated from the C-terminus was examined in E. coli culture. The C-terminal Lys316 residue was not significant in the expression, but Val315 was critical. Variants in which Val315 was substituted with fourteen amino acids were prepared. The variants substituted with hydrophobic amino acids such as Leu and Ile were almost the same as wild-type thermolysin (WT) in the expression amount, α-helix content, and stability. Variants with charged (Asp, Glu, Lys, and Arg), bulky (Trp), or small (Gly) amino acids were lower in these characteristics than WT. All variants exhibited considerably high activities (50-100% of WT) in hydrolyzing protein and peptide substrates. The expression amount, helix content, and stability of variants showed good correlation with hydropathy indexes of the amino acids substituted for Val315. Crystallographic study of thermolysin has indicated that V315 is a member of the C-terminal hydrophobic cluster. The results obtained in the present study indicate that stabilization of the cluster increases thermolysin stability and that the variants with higher stability are expressed more in the culture. Although thermolysin activity was not severely affected by the variation at position 315, the stability and specificity were modified significantly, suggesting the long-range interaction between the C-terminal region and active site.

An accurate and sensitive Coomassie Brilliant Blue G-250-based assay for protein determination.

In this protocol we present a rapid and sensitive assay for the accurate determination of protein concentration. The assay is a modification of a previous method, and measures minimum 0.2 µg protein.

The Tll0287 protein is a hemoprotein associated with the PsbA2-Photosystem II complex in Thermosynechococcus elongatus.

Cyanobacteria have multiple psbA genes encoding PsbA, the D1 reaction center protein of the Photosystem II (PSII) complex. The thermophilic cyanobacterium Thermosynechococcus elongatus has three psbA genes differently expressed depending on the environmental conditions. Among the 344 residues of PsbA, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2 and 27 between PsbA2 and PsbA3. In this study, we found a new hemoprotein that is expressed when the T. elongatus genome has only the psbA2 gene for D1. This hemoprotein was found in both the non-membrane proteins and associated to the purified PsbA2-PSII core complex. This protein could be removed by the washing of PSII with Tris-washing or CaCl2-washing. From MALDI-TOF/TOF spectrometry, N-terminal sequencing and MALDI-MS/MS analysis upon tryptic digestion, the new hemoprotein was identified to be the tll0287 gene product with a molecular mass close to 19kDa. Until now, tll0287 was registered as a gene encoding a hypothetical protein with an unknown function. From the amino acid sequence and the EPR spectrum the 5th and 6th axial ligands of the heme iron are the His145 and likely either the Tyr93, Tyr159 or Tyr165, respectively. From EPR, the heme containing Tll0287 protein associated to PsbA2-PSII corresponds to approximately 25% of the Cytc550 content whereas, from SDS page analysis, the total amount of Tll0287 with and without the heme seems almost in a stoichiometric amount with PsbA2-PSII. Homologous genes to tll0287 are found in several cyanobacteria. Possible roles for Tll0287 are suggested.

The effect of the biological variability of samples on Coomassie blue dye based fast staining for SDS-PAGE in nonfixed gels.

Fast-staining protocols based on the use of Coomassie blue dye for SDS-PAGE separated proteins, represent a quick and simple solution for protein visualization. It has been shown however, that in some cases a phenomenon of missing spots or spot discoloration may be observed in the proteome pattern when the standard fast-staining protocol is used. In this work, it is demonstrated that this occurrence is affected by the biological variability of samples, and therefore, cannot be observed in all samples. Moreover, it is demonstrated that the phenomenon is manifested exclusively in nonfixed gels, and that including a fixation step into the fast-staining protocol prevented this phenomenon. In conclusion, it has been demonstrated that standard Coomassie blue dye based fast staining for SDS-PAGE resolved proteins is affected by the biological variability of samples in nonfixed gels.