Identifying a Ubiquitinated Adaptor Protein by a Viral E3 Ligase Through Co-immunoprecipitation.

Co-immunoprecipitation is a technique widely utilized to isolate protein complexes and study protein-protein interactions. Ubiquitinated proteins could be identified by combining co-immunoprecipitation with SDS-PAGE followed by immunoblotting. In this chapter, we use Herpes Simplex Virus 1 immediate-early protein ICP0-mediated polyubiquitination of p50 as an example to describe the method to identify a ubiquitinated adaptor protein by a viral E3 ligase by co-immunoprecipitation.

Extraction and characterization of collagen and gelatin from body wall of sea cucumbers Stichopus horrens and Holothuria arenicola.

Background: Marine invertebrates, including sponges, molluscs, jellyfish, mussels, and sea cucumbers, are abundant sources of high-quality collagen and offer advantages such as availability, ease of processing, lower inflammatory response, and good metabolic compatibility. Approximately 70% of the total protein in the body wall of sea cucumbers is collagen. Gelatin is a water-soluble protein produced from heat-denatured collagen and has various industrial applications. Methods: Pepsin-solubilized collagen was extracted from the body wall of two sea cucumber Stichopus horrens and Holothuria arenicola, species found in the Oman Sea and characterized with SDS-PAGE and amino acid composition. Then gelatin was extracted from pepsin-solubilized collagen of S. horrens and some rheological properties were measured. Results: Amino acid composition and SDS-PAGE analysis showed that the collagen from both species was type I, with one α1 chain and ß chains, with molecular weights of 125 and 250 kDa, respectively. Glycine was the most abundant amino acid in the collagen from both sea cucumber species. The pepsin-soluble collagens from both species had high levels of glycine, proline, alanine, glutamic acid, and hydroxyproline. The gelatin from S. horrens had a melting point of 30 °C and displayed exceptional thermal stability, surpassing that of mammalian gelatin. Its gelling point was 5 °C, like that of cold-water fish gelatin, with a viscosity of 2.065 cp-lower than mammal gelatins. These findings suggested that collagen and gelatin from sea cucumbers could be useful in nutraceutical, pharmaceutical and cosmetic industries.

New Pipeline for Analysing Fruit Proteolytic Products Used as Digestive Health Nutraceuticals.

Proteolytic products are extensively used in the nutraceutical sector to improve protein digestion and muscle quality in target populations (e.g., athletes or elderly). These products are processed using techniques that often lead to low purity but competitive pricing. Despite their widespread use and well-established production methods, the industry lacks standardized analytical methods for assessing these products and detecting potential fraud. This study proposes a comprehensive and harmonized pipeline for their analysis, which includes quantifying total soluble protein and proteolytic activity, as well as the determination of product stability and protein profile using SDS-PAGE and proteomic techniques. Despite the fact that protease extracts from pineapple had the highest protein content, most of the bromelain remained inactive, unlike in kiwi and papaya. SDS-PAGE revealed partial protein degradation of pineapple extracts, whereas kiwi extracts reflected a lower purification level but a higher protein integrity. The application of proteomic approaches strengthened the identification and origin tracing of the proteases. This study contributes to the development of a robust framework for analyzing proteolytic extracts, spanning from soluble protein quantification to protein profiling and activity determination. It may also ensure reliable supplier selection, high-quality manufacturing practices, and the implementation of optimal storage and formulation strategies in the nutraceutical industry.

Analysis of mitochondrial translation using click chemistry.

Mitochondria contain their own gene expression machinery, which synthesizes core subunits of the oxidative phosphorylation system. Monitoring mitochondrial translation within spatial compartments of cells is difficult. Here we describe a method to visualize mitochondrial translation within defined parts of cells, using a click chemistry approach. This method can be applied to different cell types such as neurons and allows detection of newly synthesized mitochondrial proteins in spatial resolution using microscopy techniques. Furthermore, using click chemistry, mitochondrial translation can also be monitored by standard SDS-PAGE. The described method avenues the analysis of newly synthesized mitochondrial encoded proteins in the cellular context, by avoiding the usage of radioactive components.

Analysis of virulence factors in extracellular vesicles secreted by Naegleria fowleri.

Naegleria fowleri is the etiological agent of primary amebic meningoencephalitis (PAM), a rapidly progressive acute and fulminant infection that affects the central nervous system, particularly of children and young adults, which has a mortality rate greater than 95%, and its symptomatologic similarity with other meningitis caused by virus or bacteria makes it difficult to make a quick and timely diagnosis that prevents the progression of the infection. It is necessary to know the antigenic determinants as well as the pathogenicity mechanisms of this amoeba to implement strategies that allow for better antiamoebic therapeutic and diagnostic targets that directly impact the health sector. Therefore, the aim of this work was to analyze some virulence factors as part of extracellular vesicle (EV) cargo secreted by N. fowleri. The EV secretion to the extracellular medium was evaluated in trophozoites fixed and incubated with anti-N. fowleri antibody while molecular identification of EV cargo was performed by SDS-PAGE, Western blot, and RT-PCR. Our results showed that N. fowleri secretes a wide variety of vesicle sizes ranging from 0.2 to > 2 µm, and these EVs were recognized by antibodies anti-Naegleropore B, anti-19 kDa polypeptide band, anti-membrane protein Mp2CL5, anti-protease cathepsin B, and anti-actin. Furthermore, these vesicles were localized in the trophozoites cytoplasm or secreted into the extracellular medium. Specifically in relation to small vesicles, our purified exosomes were recognized by CD63 and Hsp70 markers, along with the previously mentioned proteins. RT-PCR analysis was made through the isolation of EVs from N. fowleri trophozoite culture by concentration, filtration, and ultracentrifugation. Interestingly, we obtained PCR products for Nfa1, NPB, Mp2CL5, and CatB genes as part of exosomes cargo. This suggests that the molecules identified in this work could play an important role in communication as well as in infectious processes caused by this amoeba. Therefore, the study and characterization of the pathogenicity mechanisms, as well as the virulence factors released by N. fowleri remains a key point to provide valuable information for the development of therapeutic treatments, vaccine design, or biomarkers for a timely diagnosis against infections caused by protozoa.

Revisiting Protein Reversed-Phase Chromatography for Bottom-Up Proteomics.

We revisited protein reversed-phase chromatography (RP), using state-of-the-art RP columns developed for biopharmaceuticals, such as monoclonal antibodies, in order to evaluate the suitability of this methodology as a prefractionation step for bottom-up proteomics. The protein RP prefractionation (Prot-RP) method was compared with two other widely used prefractionation methods, SDS-PAGE and high-pH peptide RP (Pept-RP) by using cell lysates as samples. The overlap between fractions of Prot-RP was comparable to that of SDS-PAGE, and the protein recovery was approximately 2-fold higher. On the other hand, the overlap between fractions of Prot-RP was slightly larger than that of Pept-RP, but Prot-RP was able to identify more protein termini and more isoform-specific peptides than Pept-RP. Our results indicate that the combination of highly efficient protein prefractionation with modern mass spectrometers is particularly effective for proteoform profiling from cellular samples.

Characterisation of seryl tRNA synthetase (srs-2) in Haemonchus contortus and Teladorsagia circumcincta.

The aim of the study was to purify and characterise recombinant proteins with the potential as an anti-parasite vaccine. Full-length cDNAs encoding seryl-tRNA synthetase (srs-2) were cloned from Haemonchus contortus (HcSRS-2) and Teladorsagia circumcincta (TcSRS-2). TcSRS-2 and HcSRS-2 cDNA (1458bp) encoded proteins of 486 amino acids, each of which was present as a single band of about 55 kDa on SDS-PAGE. Multiple alignments of the protein sequences showed homology of 94% between TcSRS-2 and HcSRS-2, 76-93% with SRS-2s of eight nematodes and 68% with Mus musculus SRS-2. The predicted three-dimensional structures revealed an overall structural homology of TcSRS-2 and HcSRS-2, highly conserved binding and catalytic sites, and minor differences in the tautomerase binding site residues in other nematode SRS-2 homologues. A phylogenetic tree was constructed using helminth and mammalian SRS-2 sequences. Soluble C-terminal SRS-2 proteins were expressed in Escherichia coli strain AY2.4 and purified. Recombinant HcSRS-2 assay shows that the recombinant enzyme was active and stable. The Km and Vmax for ATP were 3.9 ± 1.0 µM and 2.7 ± 0.1 µmol min-1 mg-1 protein, respectively. Antibodies in serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant HcSRS-2 and TcSRS-2 in enzyme-linked immunosorbent assays. Recognition of the recombinant proteins by antibodies generated by exposure of sheep to the native enzyme indicates similar antigenicity of the two proteins.

Identification and Activity Study of an Impurity Band Observed in the nrSDS-PAGE of Aflibercept.

BACKGROUND: Aflibercept is a biopharmaceutical targeting vascular endothelial growth factor (VEGF) that has shown promise in the treatment of neovascular age-related macular degeneration (nAMD) and diabetic macular edema (DME) in adults. Quality control studies of aflibercept employing non-reduced SDS-PAGE (nrSDS-PAGE) have shown that a significant variant band (IM1) is consistently present below the main band. Considering the quality control strategy of biopharmaceuticals, structural elucidation and functional studies are required. METHODS: In this study, the variant bands in nrSDS-PAGE were collected through electroelution and identified by peptide mass fingerprinting based on liquid chromatography-tandem MS (LC-MS/MS). This variant was expressed using knob-into-hole (KIH) design transient transfection for the detection of ligand affinity, binding activity and biological activity. RESULTS: The variant band was formed by C-terminal truncation at position N99 of one chain in the aflibercept homodimer. Then, this variant was successfully expressed using KIH design transient transfection. The ligand affinity of the IM1 truncated variant was reduced by 18-fold, and neither binding activity nor biological activity were detected. CONCLUSIONS: The efficacy of aflibercept is influenced by the loss of biological activity of the variant. Therefore, this study supports the development of a quality control strategy for aflibercept.

Gluten Is Not Gluten.

Wheat gluten is responsible for the unique baking properties of wheat flour, but it also causes wheat-related disorders in predisposed individuals. Different commercially available gluten materials are commonly used for a variety of assays, but a detailed characterization of their composition is missing in many cases. This is why we aimed to provide an in-depth analysis of three commonly used gliadin and gluten materials from two different batches using gel electrophoretic and chromatographic techniques. The gliadin material did not show the typical qualitative and quantitative protein composition and does not appear to be representative of wheat gliadin. The two gluten materials had the expected protein composition, but both showed large batch-to-batch variability regarding total protein content. Since these variations result in different biochemical, immunological, and functional behaviors, it is important to analyze at least the total protein content of each material and each batch.

Preparation, characterization and functional evaluation of soy protein isolate-peach gum conjugates prepared by wet heating Maillard reaction.

This study was conducted to formulate a conjugate of soy protein isolate (SPI) and peach gum (PG) with improved functional properties, interacting at mass ratios of 1:1, 1:2, 1:3, 2:1, and 2:3 by Maillard reaction via wet heating method. Conjugation efficiency was confirmed by grafting degree (DG) and browning index (BI). Results indicated that DG increased with increasing concentration of PG, and decreased with increasing pH, whereas no remarkable change was observed with increasing reaction time. The conjugates were optimized at a ratio of 1:3. SDS-PAGE confirmed conjugate formation, Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) verified conjugate secondary structural changes, and scanning electron microscopy (SEM) indicated significant overall structural changes. The functional properties, solubility, emulsifying stability, water holding, foaming, and antioxidant activity were significantly improved. This study revealed the wet heating method as an effective approach to improve the functional properties of soy protein.

Polysaccharides Analysis of Cell Wall Using Carbohydrate Gel Electrophoresis.

The plant cell wall is rich in polysaccharides with high heterogeneity. Investigating the composition and structure of cell wall polysaccharides is crucial for understanding the functionalities of plant cell walls. Carbohydrate electrophoresis is a sensitive and rapid method to analyze polysaccharides qualitatively and quantitatively. The process includes digesting the polysaccharides with appropriate cleavage enzymes, labeling the reducing ends of the released oligosaccharides with a highly charged fluorophore, and separating the labeled oligosaccharides in a polyacrylamide gel via high-voltage electrophoresis. The generated fluorescence can be calculated as compared to that of oligosaccharide standards. Therefore, this is a convenient method for polysaccharide characterization that can be performed in most laboratories. Here, we introduce the detailed operational steps and precautions, which are helpful for researchers to quickly obtain the structural information of polysaccharides.

Identification and Quantification of Extracellular Vesicles: Comparison of SDS-PAGE Analysis and Biosensor Analysis with QCM and IDT Chips.

This study presents and compares two methods for identifying the types of extracellular vesicles (EVs) from different cell lines. Through SDS-PAGE analysis, we discovered that the ratio of CD63 to CD81 in different EVs is consistent and distinct, making it a reliable characteristic for recognizing EVs secreted by cancer cells. However, the electrophoresis and imaging processes may introduce errors in the concentration values, especially at lower concentrations, rendering this method potentially less effective. An alternative approach involves the use of quartz crystal microbalance (QCM) and electroanalytical interdigitated electrode (IDT) biosensors for EV type identification and quantification. The QCM frequency shift caused by EVs is directly proportional to their concentration, while electroanalysis relies on measuring the curvature of the I-V curve as a distinguishing feature, which is also proportional to EV concentration. Linear regression lines for the QCM frequency shift and the electroanalysis curvature of various EV types are plotted separately, enabling the estimation of the corresponding concentration for an unknown EV type on the graphs. By intersecting the results from both biosensors, the unknown EV type can be identified. The biosensor analysis method proves to be an effective means of analyzing both the type and concentration of EVs from different cell lines.

Proteomic Analysis and Immunoprofiling of Persian Horned Viper Venom, Pseudocerastes Persicus, from Central Part of Iran.

Numerous species of venomous snakes of medical importance exist in Iran. Pseudocerastes persicus (P. persicus), one of the medically important snakes, also called the Persian horned viper, has a geographical spread that extends to the east, southwest, and central areas of Iran and is endemic across the wider region. As a result, this species is responsible for many snakebite occurrences. Venom from P. persicus found in the central province of Semnan contains phospholipase A2 and L-amino acid oxidase activities, and high toxic potency. The venom was fractionated by reverse-phase high-performance liquid chromatography (HPLC) and analyzed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and two-dimensional electrophoresis. Using liquid chromatography with tandem mass spectrometry (LC-MS/MS), a range of components were identified, consistent with the biochemical and toxicological properties of the venom. Proteins identified from 2D electrophoresis and shotgun methods included metallo- and serine proteases, phospholipases, oxidases, and Kunitz trypsin inhibitors, along with many other components at lower qualitative abundance. This study provides a more detailed understanding of the protein profile of Iranian P. persicus venom, which can be effective in the production of an effective antidote against it. The analysis of the resulting data shows that there is a wide range of proteins in the venom of the Persian horned viper. This information can provide a better understanding of how venom is neutralized by polyclonal antivenom. Considering the wide presence of this snake and its related species in Iran and surrounding countries, knowing the venom protein profile of this family can be of great support to antivenom producers such as Razi Vaccine & Serum Research Institute in the preparation of regional antivenoms.

The [(bathophenanthroline)3:Fe2+] complex as an aromatic non-polymeric medium for purification of human lactoferrin.

We describe a non-chromatographic, ligand-free platform for the efficient purification of recombinant human lactoferrin (LF). The platform consists of a [metal:chelator] complex precipitate in the presence of osmotically active polyethylene glycol 6000 (PEG-6000). Purification is achieved in three stages. Following formation of the complex, LF is captured under neutral conditions by the aggregated complexes (Step I), a washing step follows (Step II) and then, (Step III) LF is extracted in pure form with 100 mM tribasic Na citrate buffer (pH 7). Of the four complexes investigated, [bathophenanthroline (batho)3:Fe2+] was determined to be the most efficient. LF is recovered with high yield (∼90%, by densitometry) and purity (≥97%, by SDS polyacrylamide gel electrophoresis (SDS-PAGE)) from an artificial contamination background comprising E. coli lysate proteins. Purified LF is demonstrated to be monomeric by dynamic light scattering (DLS); to preserve its native secondary structure by circular dichroism (CD) spectroscopy; and, as apo-LF, to efficiently inhibit bacterial growth. Process yield is not affected by a 45-fold increase in LF concentration from 0.2 to 9 mg/mL. We provide evidence that protein capture relies on [cation:π] interactions between the lysine and arginine residues of LF with the fully aromatic [(batho)3:Fe2+] complexes. The use of [metal:chelator] complex aggregates is demonstrated to provide an economical and efficient avenue for LF purification.

Combining SDS-PAGE to capillary zone electrophoresis-tandem mass spectrometry for high-resolution top-down proteomics analysis of intact histone proteoforms.

Mass spectrometry (MS)-based top-down proteomics (TDP) analysis of histone proteoforms provides critical information about combinatorial post-translational modifications (PTMs), which is vital for pursuing a better understanding of epigenetic regulation of gene expression. It requires high-resolution separations of histone proteoforms before MS and tandem MS (MS/MS) analysis. In this work, for the first time, we combined SDS-PAGE-based protein fractionation (passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry, PEPPI-MS) with capillary zone electrophoresis (CZE)-MS/MS for high-resolution characterization of histone proteoforms. We systematically studied the histone proteoform extraction from SDS-PAGE gel and follow-up cleanup as well as CZE-MS/MS, to determine an optimal procedure. The optimal procedure showed reproducible and high-resolution separation and characterization of histone proteoforms. SDS-PAGE separated histone proteins (H1, H2, H3, and H4) based on their molecular weight and CZE provided additional separations of proteoforms of each histone protein based on their electrophoretic mobility, which was affected by PTMs, for example, acetylation and phosphorylation. Using the technique, we identified over 200 histone proteoforms from a commercial calf thymus histone sample with good reproducibility. The orthogonal and high-resolution separations of SDS-PAGE and CZE made our technique attractive for the delineation of histone proteoforms extracted from complex biological systems.

LC-MS characterization of N/O-glycans of α- and ß-subunits of chicken ovomucin separated by SDS-PAGE.

As the main active glycoprotein of egg white, the biological functions of chicken ovomucin α- and ß-subunit are closely related to the structure of glycans. However, the exact composition and structure of the subunit glycans are still unknown. We obtained highly pure chicken ovomucin α-subunit and ß-subunit protein bands by the strategy combined with two-step isoelectric precipitation and SDS-PAGE gel electrophoresis. The ammonia-catalyzed one-pot procedure was then used to release and capture α-and ß-subunit protein glycans with 1-phenyl- 3-Methyl-5-pyrazolone (PMP). The N/O-glycans of bis-PMP derivatives were purified and analyzed by LC-MS. More importantly, an effective dual modification was performed to accurately quantify neutral and sialylated O-glycans through methylamidation of sialic acid residues and simultaneously through carbonyl condensation reactions of reducing ends with PMP. We first showed that the α-subunit protein has only N-glycosylation modification, and the ß-subunit only O-glycosylation, a total of 22 N-glycans and 20 O-glycans were identified in the α- and ß-subunit, respectively. In addition, the complex N-glycan (47 %) and the sialylated O-glycan (77 %) are each major types of the above subunits. Such findings in this study provide a basis for studying the functional and biological activities of chicken ovomucin glycans.

Biochemical characterisation and production kinetics of high molecular-weight (HMW) putative antibacterial proteins of insect pathogenic Brevibacillus laterosporus isolates.

BACKGROUND: Bacterial genomes often encode structures similar to phage capsids (encapsulins) and phage tails which can be induced spontaneously or using genotoxic compounds such as mitomycin C. These high molecular-weight (HMW) putative antibacterial proteins (ABPs) are used against the competitive strains under natural environment. Previously, it was unknown whether these HMW putative ABPs originating from the insect pathogenic Gram-positive, spore-forming bacterium Brevibacillus laterosporus (Bl) isolates (1821L, 1951) are spontaneously induced during the growth and pose a detrimental effect on their own survival. Furthermore, no prior work has been undertaken to determine their biochemical characteristics. RESULTS: Using a soft agar overlay method with polyethylene glycol precipitation, a narrow spectrum of bioactivity was found from the precipitated lysate of Bl 1951. Electron micrographs of mitomycin C- induced filtrates showed structures similar to phage capsids and contractile tails. Bioactivity assays of cell free supernatants (CFS) extracted during the growth of Bl 1821L and Bl 1951 suggested spontaneous induction of these HMW putative ABPs with an autocidal activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of spontaneously induced putative ABPs showed appearance of ~ 30 kDa and ~ 48 kDa bands of varying intensity across all the time intervals during the bacterial growth except in the initial hours. Statistically, spontaneously induced HMW putative ABPs of Bl 1951 exhibited a significant decrease in the number of viable cells of its producer strain after 18 h of growth in liquid. In addition, a significant change in pH and prominent bioactivity of the CFS of this particular time period was noted. Biochemically, the filtered supernatant derived from either Bl 1821L or Bl 1951 maintained bioactivity over a wide range of pH and temperature. CONCLUSION: This study reports the spontaneous induction of HMW putative ABPs (bacteriocins) of Bl 1821L and Bl 1951 isolates during the course of growth with potential autocidal activity which is critically important during production as a potential biopesticide. A narrow spectrum of putative antibacterial activity of Bl 1951 precipitate was found. The stability of HMW putative ABPs of Bl 1821L and Bl 1951 over a wide range of pH and temperature can be useful in expanding the potential of this useful bacterium beyond the insecticidal value.

Sequential Double Immunoblotting with Peptide Antibodies.

Immunoblotting, also termed western blotting, is a powerful method for detection and characterization of proteins separated by various electrophoretic techniques. The combination of sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), having high separating power, immunoblotting to synthetic membranes, and detection with highly specific peptide antibodies, is especially useful for studying individual proteins in relation to cellular processes, disease mechanisms, etc. Here, we describe a protocol for the sequential detection of various forms of an individual protein using peptide antibodies, exemplified by the characterization of antibody specificity for different forms of the protein calreticulin by double SDS-PAGE immunoblotting.

Detection and Quantitation of Endogenous Membrane-Bound RAS Proteins and KRAS Mutants in Cancer Cell Lines Using 1D-SDS-PAGE LC-MS2.

In healthy cells, membrane-anchored wild-type RAS proteins (i.e., HRAS, KRAS4A, KRAS4B, and NRAS) regulate critical cellular processes (e.g., proliferation, differentiation, survival). When mutated, RAS proteins are principal oncogenic drivers in approximately 30% of all human cancers. Among them, KRAS mutants are found in nearly 80% of all patients diagnosed with RAS-driven malignancies and are regarded as high-priority anti-cancer drug targets. Due to the lack of highly qualified/specific RAS isoform and mutant RAS monoclonal antibodies, there is a vital need for an effective antibody-free approach capable of identifying and quantifying membrane-bound RAS proteins in isoform- and mutation-specific manner. Here, we describe the development of a simple antibody-free protocol that relies on ultracentrifugation to isolate the membrane fraction coupled with single-dimensional (1D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to fractionate and enrich membrane-bound endogenous RAS isoforms. Next, bottom-up proteomics that utilizes in-gel digestion followed by reversed-phase high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS2) is used for detection and relative quantitation of all wild-type RAS proteins (i.e., HRAS, KRAS4A, KRAS4B, and NRAS) and corresponding RAS mutants (e.g., G12D, G13D, G12S, G12V). Notably, this simple 1D-SDS-PAGE-HPLC-MS2-based protocol can be automated and widely applied to multiple cancer cell lines to investigate concentration changes in membrane-bound endogenous RAS proteins and corresponding mutants in the context of drug discovery.

Detection and quantification of C-terminally tagged proteins by in-gel fluorescence.

The analysis of recombinant proteins in complex solutions is often accomplished with tag-specific antibodies in western blots. Recently, I introduced an antibody-free alternative wherein tagged proteins are visualized directly within polyacrylamide gels. For this, I used the protein ligase Connectase to selectively attach fluorophores to target proteins possessing an N-terminal recognition sequence. In this study, I extend this methodology to encompass the detection and quantification of C-terminally tagged proteins. Similar to the N-terminal labeling method, this adapted procedure offers increased speed, heightened sensitivity, and an improved signal-to-noise ratio when compared to western blots. It also eliminates the need for sample-specific optimization, enables more consistent and precise quantifications, and uses freely available reagents. This study broadens the applicability of in-gel fluorescence detection methods and thereby facilitates research on recombinant proteins.