Prior to immunoaffinity and mass spectrometry, we confirm the presence and purity of proteins in cell lysates by loading them on one dimensional Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (1D SDS PAGE) of Mini-PROTEAN System (BIO-RAD Catalog# 165–8000) as per render instructions. Briefly in the procedure, 15 µl from each samples, containing equal amount of proteins mixed with loading buffer (Bio Rad catalog #161–0738) in ratio 1:1, were loaded on 12 % Mini-PROTEAN® TGX™ Precast Protein Gel (Bio Rad catalog #4561043) and run for 45 min at 200 V having 1X SDS-PAGE running buffer (Bio Rad catalog #161–0732). After the run, gel was washed with milliQ water and stained overnight in Coomassie Brilliant Blue R-250 staining solution (Bio Rad catalog #161–0436). Then gel was destained in Coomassie Brilliant Blue R-250 (Bio Rad catalog #161–0438) destaining solution until clear bands were appeared on the gel.
Coomassie brilliant blue r 250
Manufactured by Bio-Rad
Sourced in United States, Germany, Italy, United Kingdom, France, Japan, Australia
Coomassie Brilliant Blue R-250 is a protein dye used for the detection and quantification of proteins in various analytical techniques, such as gel electrophoresis and Western blotting. It is a blue dye that binds to proteins, creating a colored complex that can be visualized and measured.
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Lab products found in correlation
Gelatin, by Merck Group (9 mentions)
Bovine serum albumin (bsa), by Merck Group (7 mentions)
Protein assay kit, by Bio-Rad (6 mentions)
Mini protean tetra cell, by Bio-Rad (6 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (6 mentions)
Acetic acid, by Merck Group (6 mentions)
Laemmli sample buffer, by Bio-Rad (6 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (5 mentions)
Methanol, by Merck Group (5 mentions)
Triton x 100, by Merck Group (5 mentions)
Mini protean 2 system, by Bio-Rad (4 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (4 mentions)
β mercaptoethanol, by Merck Group (4 mentions)
Image lab software, by Bio-Rad (4 mentions)
Glycine, by Merck Group (4 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (4 mentions)
Irdye 800cw, by LI COR (4 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (4 mentions)
Precision plus protein dual color standard, by Bio-Rad (4 mentions)
348 protocols using coomassie brilliant blue r 250
1
Cell Lysate Protein Profiling by 1D SDS-PAGE
2
SDS-PAGE and Zymogram Analysis of Proteins
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The analytical polyacrylamide gel electrophoresis of proteins in the presence of sodium dodecyl sulfate (SDS-PAGE) was performed following the method of Laemmli [42] . The protein bands were visualized with Coomassie Brilliant Blue R-250 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) staining. After electrophoresis, the protein bands were stained with Coomassie Brilliant Blue R-250 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The low molecular weight markers LMW from Amersham Biosciences were used as protein markers standards.
Zymogram analysis was monitored as reported by Kubata et al. [43] . Briefly, after proteins (50 µg) were separated by SDS-PAGE, the gel was incubated in a renaturation buffer (Buffer A) for 30 min at 70 °C with gentle shaking. The treated gel was then overlaid on a pre-cast 5 mg/ml agar gel containing 10 mg/ml birchwood xylan in buffer A. The agar plates were then incubated for 15 min at room temperature. After incubation the polyacrylamide gel was removed, and the agar gel was immersed in 30 mg/ml Congo red dye for 30 min and washed with 1.5 M NaCl solution to visualize zones of clearance corresponding to xylanase activity.
Zymogram analysis was monitored as reported by Kubata et al. [43] . Briefly, after proteins (50 µg) were separated by SDS-PAGE, the gel was incubated in a renaturation buffer (Buffer A) for 30 min at 70 °C with gentle shaking. The treated gel was then overlaid on a pre-cast 5 mg/ml agar gel containing 10 mg/ml birchwood xylan in buffer A. The agar plates were then incubated for 15 min at room temperature. After incubation the polyacrylamide gel was removed, and the agar gel was immersed in 30 mg/ml Congo red dye for 30 min and washed with 1.5 M NaCl solution to visualize zones of clearance corresponding to xylanase activity.
3
Dihydrolipoamide Synthesis and Antibody Generation
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Dihydrolipoamide was synthesized from lipoamide using sodium borohydride as previously described 34 (link), 35 . All PCR primers were purchased from Life Technologies (Carlsbad, CA). ε-amino-N-caproic acid was obtained from MP Biochemicals. Acrylamide/bisacrylamide, ammonium persulfate, Bradford protein assay solution, and Coomassie brilliant blue (CBB) R-250 were from Bio-Rad laboratories (Richmond, CA, USA). NADH, BSA, lipoamide, EDTA, and NBT chloride tablets were obtained from Sigma (St. Louis, MO, USA). Serva Blue G was purchased from Serva (Heidelberg, Germany). Rabbit anti-DLDH polyclonal antibodies (IgG) and goat anti-rabbit IgG conjugated with horseradish peroxidase were purchased from US Biological (Swampscott, MA, USA) and Invitrogen (San Diego, CA, USA), respectively. Hybond-C membrane and an ECL immunochemical detection kit were obtained from GE Healthcare (Piscataway, NJ, USA).
4
Biochemical Reagent Sourcing for Protein Analysis
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Sucrose and mannitol were purchased from BDH Chemicals and Mallickrodt Chemicals, respectively. Bis-Tris, tricine, and amino-caproic acid were purchased from MB Biochemicals (Irvine, CA). Pre-stained SDS-PAGE markers were purchased from Thermo Scientific (Pittsburgh, PA). Bradford protein assay solution and Coomassie brilliant blue (CBB) R-250 were from Bio-Rad laboratories (Richmond, CA). Silver nitrate, streptozotocin (STZ), sodium citrate, NADH, EDTA, n-dodecyl-β-D-maltoside (DDM), and nitro blue tetrazolium (NBT) chloride tablets were obtained from Sigma (St. Louis, MO, USA). Serva Blue G was purchased from Serva (Heidelberg, Germany). Rabbit anti-HNE polyclonal antibodies (IgG) and goat anti-rabbit IgG conjugated with horseradish peroxidase were purchased from US Biological (Salem, MA) and Invitrogen (San Diego, CA), respectively. Hybond-C membrane and a Western blot detection kit were obtained from GE Healthcare (Piscataway, NJ).
5
Protein Extraction and Fractionation from DDGS
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Proteins before and after fermentation of the two DDGS were extracted as described by (Hamaker et al. 1995 ). Both prolamins and nonprolamins of each sample were pooled together. The concentration of protein in the samples was determined with the BCA protein assay kit (keyGEN bioTECH, Shanghai, China). Protein samples were fractionated by an SDS-PAGE system based on 12% polyacrylamide separating gels containing 0.1% SDS in Tris–glycine buffer. Approximately 6 µg of protein sample was added to each well, followed by separation at 60 mV for 210 min. The gel was stained with Coomassie Brilliant Blue (CBB) R-250 (Bio-Rad, California, USA) for 60 min and de-stained with 8% acetic acid.
6
Soluble Protein Extraction and Fractionation
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Soluble proteins in FRSM and unfermented RSM were extracted according to Faurobert [21 ] with minor modification. The samples were ground finely to pass a 60-mesh sieve. 1.5 mL of 20 mM Tris-HCl buffer (pH 7.6) including 5 μg/mL protease inhibitor, 0.1% SDS and 5 mM dithiothreitol was added to each 100 mg of the ground samples, then homogenized on ice with for 10 minutes. The homogenized samples were centrifuged at 11,000 × g for 20 minutes at 4°C (5804R, Eppendorf, Germany), then supernatants solutions were transferred to 1.5 mL Eppendorf (EP) tubes. Protein concentration in each sample was determined using Bio-Rad Protein Assay Kit (Bio-Rad, USA) according to the manufacturer's instructions. Soluble protein was fractionated by SDS-PAGE system according the previous method [22 (link)]. The electrophoresis system was based on 12% polyacrylamide separating gels containing 0.1% SDS in Tris-glycine buffer. About 20 μg of extracted protein sample was loaded for each well and separated at 65 mV for 120 minutes. After electrophoresis, the gel was stained using Coomassie Brilliant Blue (CBB) R-250 (Bio-Rad, USA) for 45 minutes and de-stained with 7% acetic acid.
7
Protein Carbonyl Assay Protocol
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Biotin-linked aldehyde reactive probe ARP) for protein carbonyl assay was from Cayman Chemical (Ann Arbor, MI). Dihydrolipoamide was synthesized from lipoamide in our own laboratory using sodium borohydride as previously described [29] , [30] . ε-amino-N-caproic acid was obtained from MP Biochemicals. Acrylamide/bisacrylamide, ammonium persulfate, Bradford protein assay solution, coomassie brilliant blue (CBB) R-250, immunoblotting membrane, and an ECL immunochemical detection kit were from Bio-Rad laboratories (Richmond, CA, USA). NADH, BSA, lipoamide, EDTA, ATP, and NBT chloride tablets were obtained from Sigma (St. Louis, MO, USA). Serva Blue G was purchased from Serva (Heidelberg, Germany). Anti-PARP antibody was purchased from Trevigen (Gaithers burg, MD). Anti-NQO1 antibodies were from Sigma. Rabbit anti-DLDH polyclonal antibodies (IgG) and goat anti-rabbit IgG conjugated with horseradish peroxidase were purchased from US Biological (Swampscott, MA, USA) and Invitrogen (San Diego, CA, USA), respectively. Other antibodies were from Abcam (Cambridge, MA).
8
Protein Purity Determination by SDS-PAGE
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Protein purity was determined using reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [19] (link) with Coomassie Brilliant Blue R-250 as the stain (Bio-Rad). Protein concentrations were determined using the Bradford protein assay (Bio-Rad, Hercules, CA) [20] (link) with bovine serum albumin as the standard.
9
Placental Protein Expression Analysis
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Western blotting was performed using placental lysate (18 µg) and STBEV (6 µg) from NP and preeclampsia placentas. STBMV and STBEX were treated with HEPES lysis buffer 1:1 (50 mmol/L HEPES, pH 7.5; 2% SDS; 10% glycerol). Western blots were incubated overnight at 4°C with mouse monoclonal anti-NEP antibody (1:1000, ab951; Abcam) and mouse monoclonal anti-PLAP (placental alkaline phosphatase) antibody (1:1000; NDOG2, in-house antibody). After incubation with HRP-conjugated anti-mouse IgG antibody (1:4000) for 1 hour at room temperature, Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) was used for chemiluminescence detection of antigens on x-ray film. Coomassie Brilliant Blue R-250 (Bio-Rad Laboratories, CA) was used to stain total protein, and bands were normalized to this using ImageJ, version 1.51 (public domain).
10
Actin Filament Depolymerization Dynamics
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5 μM G-actin was polymerized into F-actin in F-buffer (25 mM Imidazole, pH 7.8, 100 mM KCl, 2 mM MgCl2, 1 mM EGTA, 2 mM Tris–HCl, 0.2 mM CaCl2, 0.2 mM ATP, and 0.5 mM DTT) for 1 h, at room temperature. 5 μM Tpm1.1 was added to the filaments and incubated for 30 min, at room temperature. Depolymerization of F-actin in the absence or presence of Tpm1.1 was initiated by diluting actin filaments 5-fold with F-buffer in the presence of 2 μM CFL2 and differing concentrations of CAP2 (0, 0.03, 0.06, 0.125, 0.25, 0.5 μM). After incubation at room temperature for 30 min, the filaments were pelleted at 70,000 RPM (Beckman-Coulter TLA-100 Rotor), for 30 min, at room temperature. The supernatants and pellets were separated and solubilized in 1× Laemmli sample buffer for 30 min, at room temperature. The samples were boiled at 100 °C, 5 min and resolved on 11% SDS polyacrylamide gels. Gels were stained with Coomassie Brilliant Blue R-250 (Bio-Rad) and images were captured with an Epson Perfection 2450 Scanner. Quantitative analysis of the background-corrected band densities of the pellet and supernatant samples was done using ImageJ.
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