All reagents were purchased from Sigma-Aldrich (Lyon, France), unless stated otherwise. The human visfatin/Nampt enzyme-linked immunosorbent assay (ELISA) kit was from Adipogen (San Diego, CA, USA). New-born Swiss mice were from Janvier (St Berthevin, France). The anti-human visfatin/Nampt polyclonal antibody was from Alexis (Paris, France). The immunoblot nitrocellulose transfer membranes for western blot analysis were from Whatman (Dassel, Germany). The western blot enhanced chemiluminescence system and kaleidoscope prestained standards were from Bio-Rad (Marnes-la-Coquette, France). The Cyclex visfatin/Nampt colorimetric assay kit was from MBL International (Woburn, MA, USA). Recombinant mouse and human visfatin/Nampt (produced in Escherichia coli with residual lipopolysaccharide contamination <100 pg/ml according to the manufacturer) was from Alexis Biochemicals (Paris, France). APO866, a gift from Astellas Pharma (Munich, Germany), was provided by Alexander So (Rheumatology Department, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Switzerland) and also purchased from Alexis Biochemicals. IL-1β was from PeproTech (Rocky Hill, NJ, USA).
Kaleidoscope prestained standard
Manufactured by Bio-Rad
Sourced in United States, France
Kaleidoscope Prestained Standards is a set of pre-stained protein standards used for molecular weight determination in SDS-PAGE. The standards are a mixture of recombinant proteins with a broad range of molecular weights, pre-stained with a colored dye for easy visualization.
Automatically generated - may contain errors
Lab products found in correlation
Bradford assay, by Bio-Rad (2 mentions)
Total akt, by Cell Signaling Technology (2 mentions)
Pakt s473, by Cell Signaling Technology (2 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Whatman, by Cytiva (1 mentions)
Nbt bcip, by Promega (1 mentions)
Alkaline phosphatase conjugated anti mouse igg, by Merck Group (1 mentions)
Trans blot semidry apparatus, by Bio-Rad (1 mentions)
Amersham ecl kit, by GE Healthcare (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Hybond p, by GE Healthcare (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Hybond, by Cytiva (1 mentions)
Amersham ecl, by Cytiva (1 mentions)
Chemi lumi one super, by Nacalai Tesque (1 mentions)
Goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Labworks 4, by Analytik Jena (1 mentions)
Tris hcl ready gel, by Bio-Rad (1 mentions)
Endo hf, by New England Biolabs (1 mentions)
Deep purple total protein stain, by GE Healthcare (1 mentions)
19 protocols using kaleidoscope prestained standard
1
Visfatin/Nampt ELISA and Colorimetric Assay
2
Western Blot Analysis of Recombinant E. coli
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Whole-cell antigens from selected recombinant E. coli and each His-tagged protein were electrophoresed on 12% (w/v) polyacrylamide gels. The apparent molecular mass of recombinant proteins was determined using markers (kaleidoscope pre-stained standards, Bio-Rad, Hercules, CA, USA). Electrophoresed proteins were transferred to nitrocellulose membranes in a Trans-Blot-semidry-apparatus (Bio-Rad), as described by the manufacturer. Blots were incubated for 1 h at 37 °C with anti-His serum diluted 1:500, sheep sera diluted 1:100 in PBS-2% skim milk and anti-AtlA serum diluted 1:500. After several washings with phosphate-buffered saline with 2% skim milk, blots were incubated for 1 h at 37 °C with alkaline phosphatase-conjugated anti-mouse IgG (Sigma-Aldrich) diluted 1:30.000, or alkaline phosphatase-conjugated anti-sheep antibody (Sigma-Aldrich) diluted 1:10.000. After three more washes, blots were developed with bromochloroindolyl phosphate/nitroblue tetrazolium (BCIP/NBT, Promega, Madison, WI) in alkaline phosphatase buffer (100 mM NaCl, 5 mM MgCl2, 100 mM Tris, pH 9.5).
3
Western Blot Analysis of Protein Expression
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Cells (1×106) for whole cell lysate were pelleted at 700×g (5 min at 4 °C) and lysed in 50 μl of cell lysis buffer (20 mM Tris (pH 7.4), 100 mM NaCl, 1% Triton (Sigma-Aldrich), 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin and 10 μg/ml aprotinin). After 30 min on ice, the lysates were clarified by centrifugation (10 min at 4 °C) and the supernatant was collected. Protein concentration was determined by the Bradford assay (Bio-Rad, Milan, Italy). Equivalent amounts of protein were electrophoresed on SDS-PAGE gels. Kaleidoscope prestained standards (Bio-Rad) were used to determine molecular weight. The gels were then electroblotted onto PVDF membranes. After blocking with 5% milk, membranes were incubated with the primary antibody overnight. Finally, the relevant protein was visualized by staining with the appropriate secondary horseradish peroxidase-labeled antibody for 1 h followed by enhanced chemiluminescence. Densitometric analysis of the bands, relative to α-tubulin, was performed using ImageJ software (National Health Institute, Bethesda, MD, USA).
4
Western Blot Analysis of Apoptotic Proteins
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SDS polyacrylamide gel electrophoresis (SDS-PAGE) was performed using standard methods. The protein concentrations of samples were measured using a modified Lowry method (DC™ protein assay; Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of total protein (30 μg/well) from each sample were loaded onto the SDS-PAG and transferred onto polyvinylidene fluoride membranes (Hybond-P; GE, Healthcare, Little Chalfont, UK). Kaleidoscope prestained standards (Bio-Rad) were used for molecular weight calibration. Blocking was performed in 0.1% Tween 20 (v/v) and 5% nonfat milk in PBS. The membranes were incubated with anti-Bcl-2 rabbit antibody (1:1,500), anti-Bax rabbit antibody (1:1,000), anti-caspase-3 rabbit antibody (1:2,000), and anti-caspase-9 rabbit antibody (1:2,000), separately. The membranes were stripped and incubated with an anti-β-actin mouse monoclonal antibody (1:3,000) as a loading control. Expression of target protein was determined with horseradish peroxidase-conjugated antirabbit or antimouse immunoglobulin G (1:3,000) and enhanced chemiluminescence (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s suggested protocols. Kodak Xomatic AR film (Eastman Kodak, Rochester, NY, USA) was used to record the image generated by enhanced chemiluminescence using an Amersham ECL kit (GE Healthcare).
5
FABP4 Protein Expression Analysis
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In addition to the FABP4 recombinant protein (positive control), whole protein extracts (10 µg) from cultured C6 cells and mouse and rat adipose tissue, as well as excised C6 tumors from tumor-bearing mice were subjected to 5–20% SDS–polyacrylamide gel electrophoresis (E-T520L, ATTO) followed by protein transfer to PVDF membranes. For FABP4 immunological detection, a FABP4–specific monoclonal rabbit IgG (D25B3, Cell Signaling Technology, Inc.) was used with goat anti-rabbit IgG (#7074, Cell Signaling Technology, Inc.) as the secondary antibody. Detection was achieved using Chemi-Lumi One Super (Nacalai Tesque, Inc.). Blots were also incubated with a monoclonal antibody raised against β-actin, which was used as a loading control. The size of the detected proteins was estimated using Kaleidoscope Prestained Standards (Bio-Rad Laboratories, Inc.).
6
Deglycosylation of Purified PRRSV
7
Multiplex Protein Analysis using Wes-Rabbit
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Wes-Rabbit (12-230 kDa) Master Kit (# PS-MK04, # PS-MK01) including anti-rabbit secondary antibody, antibody diluent, molecular weight ladder, streptavidin-HRP, dithiothreitol (DTT), fluorescent master mix, luminol-S, peroxide, sample buffer and wash buffer were purchased from ProteinSimple (San Jose, CA, USA). This kit also provides capillary cartridge and pre-filled microplates. Antibodies used were specific for Erk1/2 (#9102), pErk1/2 (#9101), total Akt (#4691), pAktS473 (#4060) (Cell Signaling, Danvers, MA, USA) and CD36 (#sc-9154, Santa Cruz, Santa Cruz, CA, USA). Kaleidoscope pre-stained standards (#161-0324, Bio-Rad Laboratories Inc., Des Plaines, IL, USA) were used as molecular weight marker in western blot.
8
Western Blot Protein Isolation and Quantification
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Cells were rinsed in a phosphate buffer and lysed in RIPA lysis buffer to isolate proteins from PLA cells and knee joint synovium, which were then collected by centrifugation. The membrane was immersed for 1 min in ECL Advance Western Blot Detection kit (GE Healthcare, Buckinghamshire, UK) and X-ray filmed to visualize peroxidase activity and to determine the level of each protein. The intensity of each band was expressed relative to that of β-actin (1:2000, Abcam, Cambridge, UK). Kaleidoscope Prestained Standards (Bio-Rad Laboratories, Hercules, CA) were used as molecular weight markers.
9
Optimized SDS-PAGE and Native PAGE
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Protein expressions were evaluated on 10% Mini-PROTEAN TGX Precast Protein gels from Bio-Rad. For SDS-PAGE, 3 μl of a protein solution was mixed with 4 μl of 500 mM DTT and 7 μl of the 2× SDS Sample Buffer. Then, the sample was denatured at 90 °C for 5 min, snap-cooled on ice and loaded on the gel. The general running conditions for SDS were 150 V in the 1× Tris-Glycine-SDS Running Buffer from Bio-Rad. The gels were further stained with Bio-Safe Coomassie Stain from Bio-Rad. Molecular ladders used were Broad Range Protein Molecular Weight Markers from Promega and Kaleidoscope™ Prestained Standards (#161-0324 and #161-0375) from Bio-Rad. Native PAGE was performed using the same 10% Mini-PROTEAN TGX Precast Protein gels where 12 μl of protein solution was mixed with 12 μl of 2× Native Sample Buffer from Bio-Rad prior to loading (no heat denaturation employed). For Native PAGE, the molecular ladder used was NativeMark™ Unstained Protein Standard from Thermo Fisher (#LC0725).
To analyze the proteins comprising FluoroTect GreenLys, the denaturation protocol for SDS-PAGE was changed to 70 °C for 2 min, and a protein loading per well was increased to 6 μl. After the run was complete, the fluorescent images of gels were collected immediately by FluorChem Q from Alpha Innotech or a laser-based scanner Typhoon FLA 9500 from GE Healthcare, set for Alexa488 excitation and emission.
To analyze the proteins comprising FluoroTect GreenLys, the denaturation protocol for SDS-PAGE was changed to 70 °C for 2 min, and a protein loading per well was increased to 6 μl. After the run was complete, the fluorescent images of gels were collected immediately by FluorChem Q from Alpha Innotech or a laser-based scanner Typhoon FLA 9500 from GE Healthcare, set for Alexa488 excitation and emission.
10
SDS-PAGE Protein Analysis of BALF
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Fifty μg of protein mixture present in the BALF was applied in a sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) 12% slab gels according to the Laemmli method [26 (link)]; the protein bands were stained with Coomassie brilliant blue and compared to the molecular mass markers from Kaleidoscope Prestained Standards (Bio-Rad Laboratories Hercules, CA, USA).
Total protein quantity was calculated according to Bradford [27 (link)] using the Bio-Rad Protein Assay (Bio-Rad Laboratories Hercules, CA, USA) following the manufacturer's instructions. The standard curve was performed with bovine albumin solution.
Total protein quantity was calculated according to Bradford [27 (link)] using the Bio-Rad Protein Assay (Bio-Rad Laboratories Hercules, CA, USA) following the manufacturer's instructions. The standard curve was performed with bovine albumin solution.
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