The protein lysates were separated in the presence of SDS (Roth, Karlsruhe, Germany) using 10% polyacrylamide gel, according to the Laemmli method [38 (link)] and visualized with Coomassie Brilliant Blue R-250 (Roth, Karlsruhe, Germany) or transferred to the nitrocellulose membrane (Roth, Karlsruhe, Germany). The PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, Waltham, MA, USA) was used as a protein standard. The proteins fractionated by SDS-PAGE were transferred to nitrocellulose membrane [39 (link)], overlaid with anti-P1 antibodies (diluted 1:100) or Stx1B (final concentration 1 µg/mL and anti-6xHis antibody diluted 1:1000 as secondary) and anti-mouse antibody conjugated with alkaline phosphatase (diluted 1:1000) for 45 min in room temperature and visualized using NBT/BCIP reagents (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) (Roth, Karlsruhe, Germany) in 100 mM Tris pH 9.2 with the addition of 1 mM MgCl2, 1 mM MnCl2 and 150 mM NaCl.
Coomassie brilliant blue r 250
Manufactured by Carl Roth
Sourced in Germany
Coomassie Brilliant Blue R-250 is a synthetic dye used for the staining of proteins in gel electrophoresis. It is a blue dye that binds to proteins, allowing their visualization and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Carl Roth (2 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (2 mentions)
Sodium dodecyl sulfate (sds), by Carl Roth (1 mentions)
Casein, by Carl Roth (1 mentions)
α flag m2, by Merck Group (1 mentions)
Pvdf membrane, by Avantor (1 mentions)
α ubiquitin p4d1, by Santa Cruz Biotechnology (1 mentions)
Pt mr2100, by Kinematica (1 mentions)
Bovine serum albumin (bsa), by Carl Roth (1 mentions)
Chemiluminescence detection system, by GE Healthcare (1 mentions)
Rotifix, by Carl Roth (1 mentions)
Secondary horse radish peroxidase coupled antibodies, by Rockland Immunochemicals (1 mentions)
Acetic acid, by Carl Roth (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Ettan ipgphor 3 ief system, by GE Healthcare (1 mentions)
Immobiline drystrips ph 3 10, by GE Healthcare (1 mentions)
Biodocanalyze, by Analytik Jena (1 mentions)
Sonoplus, by Bandelin (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
12 protocols using coomassie brilliant blue r 250
1
SDS-PAGE and Western Blot Analysis
2
Quantifying Fungal Penetration in Arabidopsis
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Plants were grown under short-day conditions (8 h light, 22 °C; 16 h dark, 20 °C; 80 μmol m−2 s−1) and inoculated at the age of six weeks. Heavily G. orontii-infected leaves of accession Col-0 were used for leaf-to-leaf contact inoculation. Samples for microscopy were taken at 48 hpi and cleared in destaining solution (1:2 mixture of stock solution and ethanol; stock solution 1:2:1 mixture of lactic acid, glycerol and deionized H2O) at room temperature. Fungal structures were visualized with Coomassie Brilliant Blue staining (0.6% Coomassie Brilliant Blue R-250 (Carl Roth GmbH, Karlsruhe, Germany) in ethanol) by briefly dipping the specimens into the staining solution shortly prior to mounting the samples for microscopy. For quantification of fungal host cell entry, the proportion of germinated fungal sporelings that developed secondary hyphae served as an approximation of penetration success.
3
Analyzing HpHtrA Activity with Metal Ions and Inhibitors
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HpHtrA wt was incubated with 1 mM of CaCl2, MgCl2, MnCl2, ZnCl2, BaCl2, CuCl2, NiCl2, CoCl2, EDTA, or EGTA in 50 mM HEPES buffer (pH 7.4) for 60 min on ice. Non-reducing sample buffer without β-mercaptoethanol was added and the samples were separated by SDS-PAGE containing 0.1% casein (Carl Roth, Karlsruhe, Germany) as a substrate. The gel was incubated for 60 min in renaturing buffer (2.5% Triton X-100) and equilibrated for 30 min in developing buffer (50 mM Tris–HCl, pH 7.4, 200 mM NaCl, 5 mM CaCl2, and 0.02% Brij-35) at room temperature under gentle agitation. Afterwards, the gel was incubated for 24 h at 37 °C in developing buffer under gentle agitation. To visualize caseinolytic activity, the gel was stained in 0.5% Coomassie Brilliant Blue R250 (Carl Roth, Karlsruhe, Germany). For detection of multimers by SDS-PAGE, HpHtrA was separated by SDS-PAGE under non-reducing conditions. Proteins were visualized by staining with Coomassie Brilliant Blue G250. Independent experiments were repeated at least four times.
4
Immunoblotting Analysis of Ubiquitinated Proteins
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Protein extracts from total cells and eluates from immuno-precipitations were separated by standard SDS-PAGE and transferred to PVDF membranes (#10600023, VWR, USA) for the detection of Mup1-GFP, Art2-HTF and Pgk1 or to nitrocellulose membranes (#10600004, VWR, USA) for the detection of ubiquitinated Mup1. PVDF membranes were stained with Coomassie Brilliant Blue R250 (#3862.2, Roth, Germany) for assessment of transfer and loading. Antibodies used in this study include: α-FLAG M2 (#F1804, Sigma, Austria), α-GFP IgG1K (#11814460001, Sigma, Austria), α-Pgk1 22C5D8 (#459250, Invitrogen, USA), α-ubiquitin P4D1 (#3936S, Santa Cruz Biotechnology, USA). Secondary antibody was goat α-mouse IgG peroxidase (#A4416, Sigma, Austria). Western blots were developed with Advansta Western bright ECL substrate (#541005 (K-12045-D50), Biozyme, Austria).
5
Quantitative Protein Interaction Assay
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The pooled leaf sample was mixed with chilled phosphate‐buffered saline (PBS) and homogenized using a Polytron (Pt‐MR 2100, Kinematica AG, Switzerland) at maximum speed. TSP (600 μg) was incubated with 100 and 200 μg CGP. The reaction was neutralized (Law et al., 2009 ). Two samples per construct and CGP concentration were analysed. The reaction was stopped immediately or overnight using trichloroacetic acid precipitation. After centrifugation, the pellet was resolved in 300 μL 1× SDS sample buffer (Nausch et al., 2012b ), and 30 μL of the sample was analysed by 12% SDS‐PAGE. The gel was stained with Coomassie Brilliant Blue R250 (Carl Roth GmbH, Germany) for 20 min.
6
SDS-PAGE for Laccase Quantification
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Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on culture supernatants using Any kDTM precast protein gels (Biorad, USA). The supernatants of strain GSLAC was heat denatured by heating at 100 °C for 10 min in denaturing buffer containing SDS and 2-mercaptoethanol. The proteins were stained with Coomassie Brilliant Blue R-250 (Carl Roth GmbH, Germany). The amount of laccase in supernatants expressed by GSLAC was quantified using bovine serum albumin (Carl Roth GmbH, Germany) as standard.
7
Membrane Protein Expression Analysis
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Membrane fractions from Hi5 and COS7 cells, respectively, were separated on 10% SDS-polyacrylamide gels containing sodium dodecyl sulfate and either stained with Coomassie Brilliant Blue R-250 (Carl Roth, Karlsruhe, Germany) or transferred on nitrocellulose membranes. Membranes were probed with antibodies against 6xHis-tag (1:1000, RRID:AB_444306), Flag-Tag (1:1000, RRID:AB_259529), and ß-Actin (1:30,000, RRID:AB_262011) diluted in 5% skimmed milk powder in PBS + 0.3% Tween. Secondary anti-mouse antibody (RRID:AB_10015289) was diluted 1:15000.
8
Native and Plasma-Treated Ovalbumin Analysis
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All reagents, buffers, and devices were supplied by ThermoFisher Scientific unless otherwise stated. 30µl of PBS containing native or gas plasma‐treated Ova (30 µg for coomassie, 15 µg for western blot) were mixed with 4x NuPAGE LDS sample buffer and loaded without denaturation on a 10‐well 4–12% Bis‐Tris Gel. SeeBlue prestained standard was loaded, and gel electrophoresis was performed in a chamber filled with 1x MES SDS running buffer and connected to a power supply (Biometra Analytik‐Jena, Germany). For coomassie, gels were stained with 4% coomassie brilliant blue R250 in 80% methanol, 20% acetic acid (both Carl Roth, Germany), and washed with a de‐staining solution (20% methanol, 10% acetic acid, 70% ddH2O). For western blot, proteins were blotted on an activated PDVF membrane, blocked with Rotifix (Carl Roth, Germany), and stained with anti‐Ova polyclonal primary antibody (Biozol, Germany) followed by secondary horse‐radish peroxidase‐coupled antibodies (Rockland Immunochemicals). Signals were acquired after adding ECL reagent super signal WestPicoPlus in a chemiluminescence detection system (GE Healthcare, USA).
9
Quantitative Western Blot Analysis
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Western Blots were performed as described previously [90 (link)]. In total, 20 µg of whole cell protein lysates or 3 µg of histone extract were used for western blotting. β-Actin was used as housekeeper and loading control for whole cell lysates. Western blots of histone extracts were stained with Coomassie Brilliant Blue R 250 (Roth, Karlsruhe, Germany) and total H3 or H4 antibody to verify equal loading. For antibody details, see supplement (Additional file 12 : Table S2 D). The ChemiDoc Imaging System (Bio-Rad Laboratories, Feldkirchen, Germany) was used for detection. Pixel density was analyzed using the ‘Image Lab’ software provided by Bio-Rad.
10
Neutrophil Proteomic Analysis by 2DE
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Neutrophil proteins were extracted by sonication [pulse: 1 s, brake: 20 s, amplitude: 45%, on ice, Bandelin Sonoplus; Bandelin Electronic] in lysis buffer [50 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5 mM EDTA, Protease Inhibitor Cocktail] with subsequent centrifugation for pelleting cell debris as described elsewhere.21 (link) Following acetone precipitation, neutrophil proteins were separated by two-dimensional gel electrophoresis [2DE] using isoelectric focusing [IEF] dry strips [Immobiline DryStrips pH 3–10], Ettan IPGphor 3 IEF System [GE Healthcare] and followed by vertical electrophoresis with the PerfectBlue Gel System Mini L [VWR].22 (link) Semi-dry blotting to PVDF membranes [Roth] was performed with samples for immunoblotting, followed by blocking with 5% skimmed milk powder in PBS and 0.1% Tween-20 [PBST]. Membranes were incubated with serum samples diluted 1:100 in 2% skimmed milk powder in PBST for 1 h, washed with PBST and subsequently incubated with horseradish peroxidase-conjugated anti-human immunoglobulin G [IgG]. Reactive spots were analysed with a UV-transilluminator [BioDocAnalyze, Biometra] by enhanced chemiluminescence [ECL].
For spot excision and protein identification, separate 2DE gels were performed and visualized by staining with Coomassie Brilliant Blue R250 [Roth].23 (link)
For spot excision and protein identification, separate 2DE gels were performed and visualized by staining with Coomassie Brilliant Blue R250 [Roth].23 (link)
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