Antioxidant protein levels in PBMCs were determined by Western blot analysis. Twenty-microgram protein aliquots were loaded in each lane of an sodium dodecyl sulfate (SDS) polyacrylamide gel (15% acrylamide) and electrophoresed by molecular weight at 200 V for 90 min. Bands were electrotransferred onto a nitrocellulose membrane by using Trans-Blot® Turbo™ Transfer System (Bio-Rad, Segrate, Milan, Italy). The membrane was blocked (5% non-fat powdered milk in PBS, pH 7.5, containing 0.1% Tween 20) for 5 h and incubated with the corresponding primary monoclonal antibody. Antibodies anti-catalase (CAT) (1:1000, rabbit), Mn superoxide dismutase (MnSOD) (1:1000, mouse), glutathione reductase (GRd) (1:1000, mouse), glutathione peroxidase (GPx) (1:200, mouse), thioredoxin reductase 1 (TrxR1) (1:200, goat) and uncoupling protein 3 (UCP3) (1:500, mouse) were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Blots were then incubated with a secondary peroxidase-conjugated antibody (1:10,000) against specific primary antibody. Development of immunoblots was performed using an enhanced chemiluminescence kit (Immun-Star® Western C® Kit reagent, Bio-Rad Laboratories, Hercules, CA, USA). Protein bands were visualized using the image analysis program Quantity One (Bio-Rad). Precision Plus Protein Kaleidoscope™ (Bio-Rad) was used as a molecular weight marker.
Precision plus protein kaleidoscope
Manufactured by Bio-Rad
Sourced in United States
The Precision Plus Protein Kaleidoscope is a molecular weight standard for protein electrophoresis. It contains a mixture of ten recombinant proteins with molecular weights ranging from 10 kDa to 250 kDa, which can be used as a reference for determining the molecular weights of unknown protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Phenylmethanesulfonyl fluoride, by Merck Group (10 mentions)
Protease inhibitor, by Merck Group (9 mentions)
Phosphatase inhibitor, by Merck Group (9 mentions)
Luminata classico, by Merck Group (7 mentions)
Stripping solution, by Bio-Rad (6 mentions)
Crescendo western hrp substrate, by Merck Group (5 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (5 mentions)
Pierce bca protein assay reagent, by Thermo Fisher Scientific (4 mentions)
Mini protean tgx gel, by Bio-Rad (4 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (2 mentions)
Chemidoc xrs imaging system, by Bio-Rad (2 mentions)
Protran nitrocellulose transfer membrane, by Cytiva (2 mentions)
Mini protean tetra system, by Bio-Rad (2 mentions)
Gelcode blue safe protein stain, by Thermo Fisher Scientific (2 mentions)
Tris glycine sds buffer, by Bio-Rad (2 mentions)
Nanodrop 3300, by Thermo Fisher Scientific (2 mentions)
Nupage lds sample buffer 4x, by Thermo Fisher Scientific (2 mentions)
Synergy 2 machine, by Agilent Technologies (2 mentions)
Trypsin solution, by Promega (2 mentions)
45 protocols using precision plus protein kaleidoscope
1
Antioxidant Protein Levels in PBMCs
2
Electrophoretic Analysis of Antivenom Proteins
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Electrophoretic analyses of the antivenom were performed as previously described by Laemmli [33 (link)] under reducing conditions. The samples were diluted 1:1 in a sample buffer buffer (Cat # 161-0737, Bio-Rad, Hercules, CA, USA) containing β-mercaptoethanol, and were then heated at 95 °C for 5 min. Then, the samples were loaded in 12% polyacrylamide gels and were electrophoresed at 80 V for 1 h and then at 100 V for 45 min, using Tris-glycine-SDS as buffer (25 mM Tris, 192 mM glycine, pH 8.3, Bio-Rad, Cat # 161-0734). Protein bands were visualized using Coomassie stain G-250 (Bio-Rad, Cat # 161-0786). Molecular masses were determined by comparison with Precision Plus Protein Kaleidoscope (Bio-Rad, Cat # 161-0375). The antivenom profile (25 μg of protein) was compared with the profiles of 15 μg equine serum albumin (Cat # ESA-BSH, RMBIO, Missoula, MT, USA), 15 μg purified horse IgG (Fitzgerald, Cat # 31R-1055, (Cat # 31R-1055, Fitzgerald, North Acton, MA, USA), and 15 μg purified horse F(ab’)2 (Fitzgerald, Cat # 31C-CH0807).
3
SDS-PAGE Analysis of BRTF Purity
4
Production and Purification of GLP1-ELP Fusions
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The gene for each GLP1-ELP fusion, inserted under control of a T7 promoter in a pET24 plasmid, was transformed into Ultra BL21 (DE3) competent E. coli cells (Edge BioSystems). The cells were grown at 37°C in TB Dry (MO Bio) and expressed by induction of the T7 RNA polymerase with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Cells were lysed using sonication pulsed at 10 s on and 40 s off, for a total sonication time of 3 min. DNA was removed by adding 1 mL of 20 w/v% polyethylenimine (PEI) per liter culture and centrifuging at 14,000 rpm for 10 min. Fusions were subsequently purified through inverse transition cycling (ITC) by exploiting the ELP’s phase transition behavior.66 (link) Purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4–20% Tris gradient gels (Bio-Rad) negatively stained with 0.5 M CuCl2. Protein bands were compared to a standard ladder (Precision Plus Protein Kaleidoscope, Bio-Rad) to verify size.
5
Western Blot Analysis of AGT Protein
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Four micrograms of cell lysate were loaded per lane on a MiniProtean TGX™ pre-cast gel (Biorad) along with the Precision plus protein Kaleidoscope™ (BioRad) molecular mass markers. Proteins were transferred on a nitrocellulose membrane by the iBlot device (Invitrogen), and the membrane was blocked in 5% milk solution in TBST (50 mm Tris–HCl, pH 7.5, 150 mm NaCl, 0.1% Tween 20) for 1 h at RT. For AGT detection, the membrane was incubated with polyclonal rabbit anti-AGT serum (dilution 1 : 6000), washed three times in TBST and then incubated with peroxidase-conjugated anti-rabbit IgG (dilution 1 : 10 000). Blotted proteins were detected with ECL™ (Millipore), using the ChemiDocXRS Imaging System (BioRad, Hercules, CA, USA). Densitometry analysis was performed using the software ImageJ. For immunoprecipitation, 300 µg of cell lysates were incubated overnight with 300 µl of PBS buffer supplemented with 3 μg of mouse anti-FLAG IgG. The solutions were then incubated with Protein-A-Sepharose for 1 h, and the protein–antibody complexes were recovered by centrifugation. Samples were washed three times with PBS and analysed by western blot using a rabbit anti-AGT-Ma antiserum (1 : 1000).
6
Arabidopsis Thylakoid Membrane Immunoblotting
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Immunoblotting was performed as described in the protocol of BioRad laboratories. Protein samples from thylakoid membranes isolated from leaves of 5 to 6-week-old Arabidopsis plants of WT or α-CA2-KO were separated by electrophoresis in a 16% polyacrylamide gel under denaturing conditions using the Mini-PROTEAN Cell system (BioRad, Hercules, CA, USA). Precision Plus Protein Kaleidoscope (10–250 kDa) (BioRad, Hercules, CA, USA) was used as protein molecular weight markers. After electrophoresis, the proteins were transferred onto a PVDF membrane (BioRad, Hercules, CA, USA) using a wet blotting system Mini Trans-Blot Cell (BioRad, Hercules, CA, USA). Immunoblotting was carried out using primary polyclonal rabbit antibodies against D1 and PsaC proteins (Agrisera, Vännäs, Sweden) and secondary goat anti-rabbit IgG, AP conjugated antibodies (BioRad, Hercules, CA, USA). The membranes were visualized using an alkaline phosphatase conjugate substrate kit (BioRad, Hercules, CA, USA). The PVDF membranes were scanned on a flatbed scanner in transmission mode for further analysis. Quantification of the optical density of the bands on the blots was performed in the ImageJ software. The results of the Western-blot analysis were obtained from two independent experiments.
7
SDS-PAGE Protein Separation and Visualization
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Proteins were separated based
on their apparent molecular weight using a modified version of the
Laemmli SDS-PAGE17 (link) with a 5% for stacking
and 10% separating gel. 10 μg of protein from each sample was
loaded alongside a molecular-weight standard (Precision Plus Protein
Kaleidoscope, Bio-Rad). Gels were run at 94 V and were stopped when
the dye front reached ∼1 cm from the bottom.
Following
electrophoresis, the separating gel was diffusion stained (45.5% methanol,
45.5% RO H2O, 9.0% acetic acid, and 0.25% Coomassie Brilliant
Blue R250) and gently agitated overnight. The next day, the gel was
destained twice with destain 1 (45.5% methanol, 45.5% RO H2O, and 9.0% acetic acid) in 3 h intervals with agitation. Then, the
gel was placed in destain 2 overnight and agitated. Finally, the gel
was stored in 7.5% acetic acid for 1 day and scanned the following
day with an Epson Expression 1680 Scanner (Version 3.04A, Professional
Mode, Reflective, Photo Auto Exposure type, 24-bit color and 8-bit
gray-scale, Best Scanning Quality, dpi 1200, gamma 1.58).
on their apparent molecular weight using a modified version of the
Laemmli SDS-PAGE17 (link) with a 5% for stacking
and 10% separating gel. 10 μg of protein from each sample was
loaded alongside a molecular-weight standard (Precision Plus Protein
Kaleidoscope, Bio-Rad). Gels were run at 94 V and were stopped when
the dye front reached ∼1 cm from the bottom.
Following
electrophoresis, the separating gel was diffusion stained (45.5% methanol,
45.5% RO H2O, 9.0% acetic acid, and 0.25% Coomassie Brilliant
Blue R250) and gently agitated overnight. The next day, the gel was
destained twice with destain 1 (45.5% methanol, 45.5% RO H2O, and 9.0% acetic acid) in 3 h intervals with agitation. Then, the
gel was placed in destain 2 overnight and agitated. Finally, the gel
was stored in 7.5% acetic acid for 1 day and scanned the following
day with an Epson Expression 1680 Scanner (Version 3.04A, Professional
Mode, Reflective, Photo Auto Exposure type, 24-bit color and 8-bit
gray-scale, Best Scanning Quality, dpi 1200, gamma 1.58).
8
Western Blot Analysis of RBCEV Proteins
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Antibody‐conjugated RBCEVs were lysed with RIPA buffer (Thermo Fisher Scientific) containing protease inhibitors (Biotool) for 15 min on ice. Total cell lysates were extracted by incubating with RIPA buffer supplemented with protease inhibitors for 30 min on ice. A total of 100 μg protein from RBCEVs and 30 μg protein from cell lysates were loaded in 10% polyacrylamide gels along with a protein ladder (Precision Plus Protein™ Kaleidoscope, Bio‐Rad), followed by a transfer to Immobilon‐P polyvinylidene difluoride membrane (Merck Millipore). The membrane was blocked with 5% milk in 1× Tris‐buffered saline containing 0.1% Tween‐20 (TBS‐T) at room temperature for 2 h followed by an incubation with primary antibodies: rabbit anti‐FLT3 (CST), mouse anti‐GAPDH (A01020, Abbkine, USA), Pierce™ High Sensitivity Streptavidin‐HRP (Thermo Fisher Scientific) overnight at 4°C. We washed the blots 3 times with TBS‐T, and then incubated them with HRP‐conjugated anti‐mouse and anti‐rabbit secondary antibodies (Santa Cruz, USA) for 1 h at room temperature. The blots were imaged using the Bio‐Rad Chemidoc gel documentation system.
9
SDS-PAGE and Western Blot for Hrp1 Protein
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Purified protein samples were adjusted to 2% SDS, 140 mM β-mercaptoethanol, 10% glycerol, 0.002% bromophenol blue, 80 mM Tris, pH 6.8, incubated at room temperature for 10 min. (unless otherwise indicated), and separated by electrophoresis in agarose gels (1.5% w/v) in 40 mM Tris-acetate, pH 7.8; 1 mM EDTA, 0.1% SDS run at 4°C. Bio-Rad Precision Plus Protein Kaleidoscope (Cat. No. 161–0375) molecular weight markers were run as standards. Proteins were blotted to Protran nitrocellulose transfer membrane (Whatman) by capillary action for 18 hrs. Filters were blocked in 5% (w/v) nonfat dry milk in Tris-buffered saline with 0.1% Tween-20, probed with anti-Hrp1 antibody, and detected with chemiluminescence as described above.
10
Enzyme Homogeneity and Molecular Mass Determination
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Non-denaturing PAGE (12%) was used to determine homogeneity of the enzyme according to Martinez et al. [13 (link)]. SDS-PAGE and 2D gel electrophoresis were used to determine the molecular mass of the purified enzyme produced in different conditions under denaturing conditions using 12% acrylamide gel, as described by Laemmli [12 (link)]. Staining was carried out with Coomassie Brilliant Blue G-250 and the extracellular protein bands were compared to the Precision PlusProtein Kaleidoscope standards (Bio-Rad) to determine molecular masses. To identify the amylase activity ‘in gel’, 1% (w/v) starch-polymerized non-denaturing PAGE gels were flooded with 0.1 M phosphate-citrate and 0.05 M NaCl buffer (pH 6) and incubated at 39°C for 2 hours. The band with amylase activity was exposed by staining with Lugol’s solution (0.67% KI and 0.33% I2).
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