A new trypsin inhibitor from Centrosema plumieri effective against digestive proteases from Tribolium castaneum, an eco-friendly alternative.

Tribolium castaneum, also known as the red flour beetle, is a polyphagous pest that seriously damages agricultural products, including stored and processed grains. Researchers have aimed to discover alternative pest control mechanisms that are less harmful to the ecosystem than those currently used. We conduct the purification and characterization of a protease inhibitor from C. plumieri seeds and an in vitro evaluation of its insecticidal potential against the insect pest T. castaneum. The trypsin inhibitor was isolated from C. plumieri seeds in a single-step DEAE-Sepharose column chromatography and had a molecular mass of 50 kDA. When analyzed for interaction with different proteolytic enzymes, the inhibitor exhibited specificity against trypsin and no activity against other serine proteases such as chymotrypsin and elastase-2. The isolated inhibitor was able to inhibit digestive enzymes of T. castaneum from extracts of the intestine of this insect. Therefore, we conclude that the new protease inhibitor, specific in tryptic inhibition, of protein nature from the seeds of C. plumieri was effective in inhibiting the digestive enzymes of T. castaneum and is a promising candidate in the ecological control of pests.

Sulfated Aeruginosins from Lake Kinneret: Microcystis Bloom, Isolation, Structure Elucidation, and Biological Activity.

Aeruginosins are common metabolites of cyanobacteria. In the course of re-isolation of the known aeruginosins KT608A and KT608B for bioassay studies, we isolated three new sulfated aeruginosins, named aeruginosins KT688 (1), KT718 (2), and KT575 (3), from the extract of a Microcystis cell mass collected during the 2016 spring bloom event in Lake Kinneret, Israel. The structures of the new compounds were established on the basis of analyses of the 1D and 2D NMR, as well as HRESIMS data. Marfey's method, coupled with HR ESI LCMS and chiral HPLC, was used to establish the absolute configuration of the amino acid and hydroxyphenyl lactic acid residues, respectively. Compounds 1-3 were tested for inhibition of the serine protease trypsin, and compounds 1 and 2 were found to exhibit IC50 values of 2.38 and 1.43 µM, respectively.

Enrichment of Trypsin Inhibitor from Soybean Whey Wastewater Using Different Precipitating Agents and Analysis of Their Properties.

Recovering valuable active substances from the by-products of agricultural processing is a crucial concern for scientific researchers. This paper focuses on the enrichment of soybean trypsin inhibitor (STI) from soybean whey wastewater using either ammonium sulfate salting or ethanol precipitation, and discusses their physicochemical properties. The results show that at a 60% ethanol content, the yield of STI was 3.983 mg/mL, whereas the yield was 3.833 mg/mL at 60% ammonium sulfate saturation. The inhibitory activity of STI obtained by ammonium sulfate salting out (A-STI) was higher than that obtained by ethanol precipitation (E-STI). A-STI exhibited better solubility than E-STI at specific temperatures and pH levels, as confirmed by turbidity and surface hydrophobicity measurements. Thermal characterization revealed that both A-STI and E-STI showed thermal transition temperatures above 90 °C. Scanning electron microscopy demonstrated that A-STI had a smooth surface with fewer pores, while E-STI had a rough surface with more pores. In conclusion, there was no significant difference in the yield of A-STI and E-STI (p < 0.05); however, the physicochemical properties of A-STI were superior to those of E-STI, making it more suitable for further processing and utilization. This study provides a theoretical reference for the enrichment of STI from soybean whey wastewater.

A Novel Kunitz Trypsin Inhibitor from Enterolobium gummiferum Seeds Exhibits Antibiofilm Properties against Pathogenic Yeasts.

Plant peptidase inhibitors play crucial roles in plant defence mechanisms and physiological processes. In this study, we isolated and characterised a Kunitz trypsin inhibitor from Enterolobium gummiferum seeds named EgPI (E. gummiferum peptidase inhibitor). The purification process involved two chromatography steps using size exclusion and hydrophobic resins, resulting in high purity and yield. EgPI appeared as a single band of ~20 kDa in SDS-PAGE. Under reducing conditions, the inhibitor exhibited two polypeptide chains, with 15 and 5 kDa. Functional characterisation revealed that EgPI displayed an inhibition stoichiometry of 1:1 against trypsin, with a dissociation constant of 8.4 × 10-9 mol·L-1. The amino-terminal sequencing of EgPI revealed the homology with Kunitz inhibitors. Circular dichroism analysis provided insights into the secondary structure of EgPI, which displayed the signature typical of Kunitz inhibitors. Stability studies demonstrated that EgPI maintained the secondary structure necessary to exhibit its inhibitory activity up to 70 °C and over a pH range from 2 to 8. Microbiological screening revealed that EgPI has antibiofilm properties against pathogenic yeasts at 1.125 µmol·L-1, and EgPI reduced C. albicans biofilm formation by 82.7%. The high affinity of EgPI for trypsin suggests potential applications in various fields. Furthermore, its antibiofilm properties recommended its usefulness in agriculture and antimicrobial therapy research, highlighting the practical implications of our research.

Structural insights and molecular dynamics into the inhibitory mechanism of a Kunitz-type trypsin inhibitor from Tamarindus indica L.

Trypsin inhibitors from tamarind seed have been studied in vitro and in preclinical studies for the treatment of obesity, its complications and associated comorbidities. It is still necessary to fully understand the structure and behaviour of these molecules. We purifed this inhibitor, sequenced de novo by MALDI-TOF/TOF, performed its homology modelling, and assessed the interaction with the trypsin enzyme through molecular dynamics (MD) simulation under physiological conditions. We identified additional 75 amino acid residues, reaching approximately 72% of total coverage. The four best conformations of the best homology modelling were submitted to the MD. The conformation n°287 was selected considering the RMSD analysis and interaction energy (-301.0128 kcal.mol-1). Residues Ile (54), Pro (57), Arg (59), Arg (63), and Glu (78) of pTTI presented the highest interactions with trypsin, and arginine residues were mainly involved in its binding mechanism. The results favour bioprospecting of this protein for pharmaceutical health applications.

Identification of sustainable trypsin active-site inhibitors from Nigrospora sphaerica strain AVA-1.

Trypsin is a protein-digesting enzyme that is essential for the growth and regeneration of bone, muscle, cartilage, skin, and blood. The trypsin inhibitors have various role in diseases such as inflammation, Alzheimer's disease, pancreatitis, rheumatoid arthritis, cancer prognosis, metastasis and so forth. From 10 endophytic fungi isolated, we were able to screen only one strain with the required activity. The fungus with activity was obtained as an endophyte from Dendrophthoe falcata and was later identified as Nigrospora sphaerica. The activity was checked by enzyme assays using trypsin. The fungus was fermented and the metabolites were extracted and further purified by bioassay-guided chromatographic methods and the compound isolated was identified using gas chromatography-mass spectrometry. The compound was identified as quercetin. Docking studies were employed to study the interaction. The absorption, distribution, metabolism, and excretion analysis showed satisfactory results and the compound has no AMES and hepatotoxicity. This study reveals the ability of N. sphaerica to produce bioactive compound quercetin has been identified as a potential candidate for trypsin inhibition. The present communication describes the first report claiming that N. sphaerica strain AVA-1 can produce quercetin and it can be considered as a sustainable source of trypsin active-site inhibitors.

Trypsin inhibitor content and activity of soaking water whey as waste in soy milk processing.

Soybean soaking water whey (SWW) is obtained as the waste of soy milk production and mostly represents an environmental problem. The aim of this study was to assess the content of proteins and content and activity of trypsin inhibitors of fresh SWW, obtained during soy milk production. Two zones of Bowman-Birk trypsin inhibitors (BBI) were detected. One was identified as a monomeric form of BBI (0.61-2.93%) and the other one was identified as a polymeric form of BBI (0.45-3.33%). The degree of BBI extraction (1.88-5.49%) was influenced by the soybean genotype and the grain size, i.e. it increased with increasing grain size. Kunitz trypsin inhibitor was not detected. Total proteins were found in traces in SWW (0.03-0.06%). Low residual trypsin inhibitor activity (0.32-0.55%) suggested that SWW can potentially be applied for preparing food or feed. In that case it will not be waste but a cheap functional supplement with BBI as a biologically active component.

Characterization of a new member of kunitz-type protein family from the venom of Persian false-horned viper, Pseudocerastes persicus.

A new member of kunitz-type protein family, PPTI (PseudocerastesPersicusTrypsin Inhibitor), was isolated from the venom of Persian false horned viper Pseudocerastes persicus and characterized. Mass spectrometry and amino acid sequencing revealed that PPTI is a 68 amino acid protein with molecular weight of about 7.6 kDa. The first amino acid residue of PPTI is N-terminally blocked via a post translational modification to pyroglutamyl. Sequence comparison against UniProtKB shows a high sequence similarity of PPTI with kunitz-type proteins, especially serine protease inhibitors and dendrotoxins (DTXs). The number of cysteines and disulfide bonding pattern of PPTI are the same as kunitz-type proteins. Based on sequence derive information, anti-protease activity of PPTI against trypsin was experimentally examined. The constructed homology models of PPTI confirmed the ability of PPTI to fold similarly to kunitz domain. The presence of characteristic basic-hydrophobic functional dyad of DTXs in PPTI supports its inhibitory potential against potassium channels. In summary, this study hypothesized the dual functionality of PPTI according to its inhibitory effect on trypsin and its potential ability in blocking potassium channel.

Cloning of a novel trypsin inhibitor from the Traditional Chinese medicine decoction pieces, Radix Trichosanthis.

Most herbs of traditional Chinese medicine (TCM) are used as air-dried decoction pieces that are manufactured and kept at ambient temperature for long periods. Given the ability of some desiccation-tolerant plants to conserve RNA, it could be worthwhile to isolate mRNA from TCM decoction pieces as part of a transcriptomic strategy to identify new substances with potential pharmaceutical application. Here, we report the molecular cloning of a novel trypsin inhibitor (as the probable alleleic variants TKTI-2 and TKTI-3) from the decoction piece of Radix Trichosanthis, representing the dried root of Trichosanthes kirilowii. From this material, the total RNA was extracted and a cDNA library was constructed from the isolated mRNA from which the cDNAs of two precursors were successfully cloned and sequenced. TKTI-3 showed an amino-acid substitution in the otherwise highly-conserved P1-P1' reaction site of the mature peptide, which we confirmed to not be an artefact. Subsequent analysis using LC-MS/MS detected the presence of specific tryptic peptides expected from TKTI-3, confirming the presence and expression of this locus in Radix Trichosanthis. More generally, this study indicates that mRNA can persist in decoction pieces and so could present a viable option for the molecular cloning from other TCMs.

Biochemical characterization of a Kunitz inhibitor from Inga edulis seeds with antifungal activity against Candida spp.

We describe the characterization of IETI, the first trypsin inhibitor purified from Inga edulis, a tree widely distributed in Brazil. Two-step chromatography was used to purify IETI, a protein composed of a single peptide chain of 19,685.10 Da. Amino-terminal sequencing revealed that IETI shows homology with the Kunitz family, as substantiated by its physical-chemical features, such as its thermal (up to 70 °C) and wide-range pH stability (from 2 to 10), and the value of its dissociation constant (6.2 nM). IETI contains a single reactive site for trypsin, maintained by a disulfide bridge; in the presence of DTT, its inhibitory activity was reduced in a time- and concentration-dependent manner. IETI presented activity against Candida ssp., including C. buinensis and C. tropicalis. IETI inhibitory activity triggered yeast membrane permeability, affecting cell viability, thus providing support for the use of IETI in further studies for the control of fungal infections.

Discovery and Characterization of Cyclic and Acyclic Trypsin Inhibitors from Momordica dioica.

Momordica trypsin inhibitors (TIs) such as those isolated from the seeds of the gac fruit, Momordica cochinchinensis (MCoTI-I and MCoTI-II), are widely used as scaffolds for drug design studies. To more effectively exploit these molecules in the development of therapeutics, there is a need for wider discovery of the natural sequence diversity among TIs from other species in the Momordica subfamily. Here we report the discovery of the encoding gene and six TIs from the seeds of the spiny gourd, Momordica dioica, four of which possess novel sequences (Modi 1, 3, 5, and 6) and two (Modi 2 and 4) of which are known peptides (TI-14, TI-17) previously identified in Momordica subangulata. Modi 6 is an acyclic peptide featuring a pyrrolidone carboxylic acid modification, whereas the remaining five TIs are cyclic. All Modi peptides display similar overall structures and trypsin inhibitory activities. No toxicity was observed for these peptides when tested against cancer and insect cells. All Modi peptides were exceptionally stable over 24 h in human serum, indicating a dual strategy to stabilize the peptides in nature, either head-to-tail cyclization or N-pyrolation, which suggests these peptides might be excellent candidates as scaffolds for epitope stabilization in drug design studies.

LUTI: a double-function inhibitor isolated from naked flax seeds.

Acute glucose fluctuation during the postprandial period causes a risk for type 2 diabetes mellitus (T2DM). α-Glucosidase inhibitors have been approved as therapeutic agents for diabetes. In the present study, a protein with α-glucosidase inhibitory activity from Flax (Linum usitatissimum) seeds was isolated using a one-step purification with Q-Sepharose4B column, followed by Sephacryl S-200 size-exclusion chromatography. It was identified as a trypsin inhibitor, named L. usitatissimum trypsin inhibitor (LUTI). The half maximal inhibitory concentration (IC50) of LUTI was 113.92 µM for α-glucosidase and 6.17 µM for trypsin. Lineweaver-Burk kinetic experiment showed that the protein exhibited two distinct inhibitory modes, a competitive inhibitor type for α-glucosidase and a non-competitive type for trypsin. The interaction between LUTI and α-glucosidase was detected through gel filtration chromatography and dynamic light scattering. Increased glucose consumption and lactic acid production were also observed following LUTI treatment in Caco-2 and HepG2 cells. LUTI inhibits not only the activity of trypsin but also the activity of α-glucosidase. It is expected that LUTI will become an oral hypoglycemic polypeptide drug for T2DM.

A Versatile and Robust Serine Protease Inhibitor Scaffold from Actinia tenebrosa.

Serine proteases play pivotal roles in normal physiology and a spectrum of patho-physiological processes. Accordingly, there is considerable interest in the discovery and design of potent serine protease inhibitors for therapeutic applications. This led to concerted efforts to discover versatile and robust molecular scaffolds for inhibitor design. This investigation is a bioprospecting study that aims to isolate and identify protease inhibitors from the cnidarian Actinia tenebrosa. The study isolated two Kunitz-type protease inhibitors with very similar sequences but quite divergent inhibitory potencies when assayed against bovine trypsin, chymostrypsin, and a selection of human sequence-related peptidases. Homology modeling and molecular dynamics simulations of these inhibitors in complex with their targets were carried out and, collectively, these methodologies enabled the definition of a versatile scaffold for inhibitor design. Thermal denaturation studies showed that the inhibitors were remarkably robust. To gain a fine-grained map of the residues responsible for this stability, we conducted in silico alanine scanning and quantified individual residue contributions to the inhibitor's stability. Sequences of these inhibitors were then used to search for Kunitz homologs in an A. tenebrosa transcriptome library, resulting in the discovery of a further 14 related sequences. Consensus analysis of these variants identified a rich molecular diversity of Kunitz domains and expanded the palette of potential residue substitutions for rational inhibitor design using this domain.

A New Approach of Extraction of α-Amylase/trypsin Inhibitors from Wheat (Triticum aestivum L.), Based on Optimization Using Plackett-Burman and Box-Behnken Designs.

Wheat is one of the most consumed foods in the world and unfortunately causes allergic reactions which have important health effects. The α-amylase/trypsin inhibitors (ATIs) have been identified as potentially allergen components of wheat. Due to a lack of data on optimization of ATI extraction, a new wheat ATIs extraction approach combining solvent extraction and selective precipitation is proposed in this work. Two types of wheat cultivars (Triticum aestivum L.), Julius and Ponticus were used and parameters such as solvent type, extraction time, temperature, stirring speed, salt type, salt concentration, buffer pH and centrifugation speed were analyzed using the Plackett-Burman design. Salt concentration, extraction time and pH appeared to have significant effects on the recovery of ATIs (p < 0.01). In both wheat cultivars, Julius and Ponticus, ammonium sulfate substantially reduced protein concentration and inhibition of amylase activity (IAA) compared to sodium chloride. The optimal conditions with desirability levels of 0.94 and 0.91 according to the Doehlert design were: salt concentrations of 1.67 and 1.22 M, extraction times of 53 and 118 min, and pHs of 7.1 and 7.9 for Julius and Ponticus, respectively. The corresponding responses were: protein concentrations of 0.31 and 0.35 mg and IAAs of 91.6 and 83.3%. Electrophoresis and MALDI-TOF/MS analysis showed that the extracted ATIs masses were between 10 and 20 kDa. Based on the initial LC-MS/MS analysis, up to 10 individual ATIs were identified in the extracted proteins under the optimal conditions. The positive implication of the present study lies in the quick assessment of their content in different varieties especially while considering their allergenic potential.

Protective effect of a protease inhibitor from Agaricus bisporus on Saccharomyces cerevisiae cells against oxidative stress.

Protease inhibitors are known to resist damage to host organisms against external threats, hence form a part of their defense system. This property of protease inhibitors was studied on protecting oxidatively stressed Saccharomyces cerevisiae yeast cells. The protease inhibitor was extracted from Agaricus bisporus, an edible mushroom. The inhibitor showed the presence of antioxidant activity as the purified inhibitor fraction gave an IC50 value of 45.13 ± 0.88 µg/mL and 33.30 ± 1.5 µg/mL when checked, respectively, by 2, 2-diphenyl-1-picrylhydrazyl, DPPH and 2, 2'-azo-bis(3-ethylbenzthiazoline-6- sulfonic acid), ABTS•+ scavenging activity. The yeast cells' survival rate (%), was determined through 3-(4, 5-dimethylthiazol-2-yl) - 2, 5-diphenyltetrazolium bromide, MTT assay, and it was found that in the presence of 2 mM H2O2 cell survival decreased to 26.33%, whereas when the experiment was conducted in the presence of protease inhibitor and 2 mM H2O2 cell survival percentage rose to 74%. The protease inhibitor's effect on the oxidatively stressed yeast cells was further studied by using Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) and Confocal Microscopy to understand the morphological changes. The viable and non-viable cell populations were quantified using Fluorescence Assorted Cell Sorting (FACS) using propidium iodide, PI, 4', 6-diamidino-2-phenylindole, DAPI and 2', 7'-dichlorofluorescein, DCF dyes.

Dual Insecticidal Effects of Adenanthera pavonina Kunitz-Type Inhibitor on Plodia interpunctella is Mediated by Digestive Enzymes Inhibition and Chitin-Binding Properties.

The Indianmeal moth, Plodia interpunctella, is one of the most damaging pests of stored products. We investigated the insecticidal properties of ApKTI, a Kunitz trypsin inhibitor from Adenanthera pavonina seeds, against P. interpunctella larvae through bioassays with artificial diet. ApKTI-fed larvae showed reduction of up to 88% on larval weight and 75% in survival. Trypsin enzymes extracted from P. interpunctella larvae were inhibited by ApKTI, which also demonstrated capacity to bind to chitin. Kinetic studies revealed a non-competitive inhibition mechanism of ApKTI for trypsin, which were further corroborated by molecular docking studies. Furthermore, we have demonstrated that ApKTI exhibits a hydrophobic pocket near the reactive site loop probably involved in chitin interactions. Taken together, these data suggested that the insecticidal activity of ApKTI for P. interpunctella larvae involves a dual and promiscuous mechanisms biding to two completely different targets. Both processes might impair the P. interpunctella larval digestive process, leading to larvae death before reaching the pupal stage. Further studies are encouraged using ApKTI as a biotechnological tool to control insect pests in field conditions.

Cloning and characterization of serpin from red king crab Paralithodes camtschaticus.

Serpins are a family of serine protease inhibitors that are involved in numerous physiological processes and are known to regulate innate immunity pathways. To advance our understanding of their role in P. camtschaticus, a commercially significant species, we cloned and characterized a serpin from this species, designated serpin PC, that has anticoagulant and anticomplement effects on human blood. We found that serpin PC is a secreted protein with a typical serpin-like primary structure that is similar to other known crustacean serpins. Recombinant serpin PC was found to have inhibitory activity against R/K-specific bovine cationic trypsin. The reaction proceeds through the formation of a stable covalent complex of peptidase with P1 residue R383 of serpin PC. This interaction is characterized by a relatively high overall inhibition constant kass=(2.3 ±â€¯0.7) × 106 M-1s-1 and an SI of 4.7 ±â€¯0.8. Protein localization by western blotting showed that serpin PC is present in the muscles and, to a lesser extent, the heart, whereas it is transcribed predominantly in hemocytes and the heart. Through peptidase activity profiling of hemocytes and plasma, we found that serpin PC inhibits at least two R/K-specific activities and showed that it inhibits phenoloxidase (PO) activity induction in hemocytes.

A Bowman-Birk Inhibitor from the Seeds of Luetzelburgia auriculata Inhibits Staphylococcus aureus Growth by Promoting Severe Cell Membrane Damage.

Staphylococcus aureus is a multidrug-resistant bacterium responsible for several cases of hospital-acquired infections, which constitute a global public health problem. The introduction of new healthcare strategies and/or the discovery of molecules capable of inhibiting the growth or killing S. aureus would have a huge impact on the treatment of S. aureus-mediated diseases. Herein, a Bowman-Birk protease inhibitor ( LzaBBI), with strong in vitro antibacterial activity against S. aureus, was purified to homogeneity from Luetzelburgia auriculata seeds. LzaBBI in its native form is a 14.3 kDa protein and has a pI of 4.54, and its NH2-terminal sequence has high identity with other Bowman-Birk inhibitors. LzaBBI showed a mixed-type inhibitory activity against both trypsin and chymotrypsin, respectively, and it remained stable after both boiling at 98 °C for 120 min and incubation at various pHs. Scanning electron microscopy revealed that LzaBBI disrupted the S. aureus membrane integrity, leading to bacterial death. This study suggests that LzaBBI is a powerful candidate for developing a new antimicrobial to overcome drug resistance toward reducing hospital-acquired infections caused by S. aureus.

Physico-Chemical and Antifungal Properties of a Trypsin Inhibitor from the Roots of Pseudostellaria heterophylla.

Plant peptidase inhibitors play essential roles in the defense systems of plants. A trypsin inhibitor (PHTI) with a molecular mass of 20.5 kDa was isolated from the fresh roots of the medicinal herb, Pseudostellaria heterophylla. The purification process involved ammonium sulfate precipitation, gel filtration chromatography on Sephadex G50, and ion-exchange chromatography on DEAE 650M. The PHTI contained 3.7% α-helix, 42.1% ß-sheets, 21.2% ß-turns, and 33% disordered structures, which showed similarity with several Kunitz-type trypsin inhibitors. Inhibition kinetic studies indicated that PHTI was a competitive inhibitor, with a Ki value of 3.01 × 10-9 M, indicating a high affinity to trypsin. The PHTI exhibited considerable stability over a broad range of pH (2⁻10) and temperatures (20⁻70 °C); however, metal ions, including Fe3+, Ba2+, Mn2+, and Al3+, could inactivate PHTI to different degrees. Results of fluorescence spectroscopy and circular dichroism showed that Fe3+ could bind to TI with an association constant of 2.75 × 105 M-1 to form a 1:1 complex, inducing conformation changes and inactivation of PHTI. In addition, PHTI could inhibit the growth of the phytopathogens, Colletotrichum gloeosporioides and Fusarium oxysporum, through disruption of the cell membrane integrity. The present study extended research on Pseudostellaria heterophylla proteins and makes PHTI an exploitable candidate as an antifungal protein for further investigation.

Fabrication of an Open Microfluidic Device for Immunoblotting.

Given the wide adoption of polydimethylsiloxane (PDMS) for the rapid fabrication of microfluidic networks and the utility of polyacrylamide gel electrophoresis (PAGE), we develop a technique for fabrication of PAGE molecular sieving gels in PDMS microchannel networks. In developing the fabrication protocol, we trade-off constraints on materials properties of these two polymer materials: PDMS is permeable to O2 and the presence of O2 inhibits the polymerization of polyacrylamide. We present a fabrication method compatible with performing PAGE protein separations in a composite PDMS-glass microdevice, that toggles from an "enclosed" microchannel for PAGE and blotting to an "open" PA gel lane for immunoprobing and readout. To overcome the inhibitory effects of O2, we coat the PDMS channel with a 10% benzophenone solution, which quenches the inhibiting effect of O2 when exposed to UV, resulting in a PAGE-in-PDMS device. We then characterize the PAGE separation performance. Using a ladder of small-to-mid mass proteins (Trypsin Inhibitor (TI); Ovalbumin (OVA); Bovine Serum Albumin (BSA)), we observe resolution of the markers in <60 s, with separation resolution exceeding 1.0 and CVs of 8.4% for BSA-OVA and 2.4% for OVA-TI, with comparable reproducibility to glass microdevice PAGE. We show that benzophenone groups incorporated into the gel through methacrylamide can be UV-activated multiple times to photocapture protein. PDMS microchannel network is reversibly bonded to a glass slide allowing direct access to separated proteins and subsequent in situ diffusion-driven immunoprobing and total protein Sypro red staining. We see this PAGE-in-PDMS fabrication technique as expanding the application and use of microfluidic PAGE without the need for a glass microfabrication infrastructure.