Tissues or cells were lysed with ice-cold RIPA buffer [50 mM Tris-HCl (pH7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxysodium cholate, 0.1% SDS, 5 mM EDTA, 10 mM NaF. Before use, add 1 mM PMSF, 3 mM dithiothreitol, 1 mM sodium vanadate, and protease inhibitors (Merck)]. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes or polyvinylidene fluoride membranes. Immunoblots were developed in chemiluminescence reagent (PerkinElmer Life Sciences) and exposed in a Fujifilm LAS 4000 imager.
Chemiluminescence reagent
Manufactured by PerkinElmer
Sourced in United States
Chemiluminescence reagent is a laboratory product that generates light through a chemical reaction, which can be used for various analytical and detection purposes. The core function of this reagent is to produce light signals that can be measured and analyzed, enabling researchers to detect and quantify specific analytes in their samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by GE Healthcare (5 mentions)
Anti gapdh, by Santa Cruz Biotechnology (4 mentions)
Sds page gel, by Bio-Rad (4 mentions)
Image lab quantification software, by Bio-Rad (4 mentions)
Fusion sl, by Avantor (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Peroxidase conjugated secondary antibody, by Vector Laboratories (3 mentions)
Immobilon membrane, by Merck Group (3 mentions)
Immobilon p, by Merck Group (2 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Ab175402, by Abcam (2 mentions)
Costar spin x centrifuge tube filter, by Corning (2 mentions)
Ab113691, by Abcam (2 mentions)
Ab124680, by Abcam (2 mentions)
Ls c197243, by LifeSpan BioSciences (2 mentions)
Stainless steel beads for tissue homogenization, by Next Advance (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Mab1594, by Merck Group (2 mentions)
Anti cugbp1, by Santa Cruz Biotechnology (2 mentions)
52 protocols using chemiluminescence reagent
1
Western Blot Protein Analysis
2
Barium Citrate Precipitation for Plasma Protein Analysis
3
Western Blot Analysis of Protein Expression
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Cells were lysed on ice in RIPA buffer (10 mM Tris pH 8.0, 150 mM NaCl, 1%NP-40, 0.5 % sodium deoxycholate, 0.1 % SDS, 5 mM EDTA) containing protease inhibitors (aprotinin, leupeptin, pepstatin, PMSF) and phosphatase inhibitors (NaF, Na3VO4). After rocking for 1 h at 4 °C, samples were centrifuged at 100,000 x g for 30 min, and the supernatants used as cell lysates. The protein content in cell lysates was estimated with the bicinchoninic acid assay (Pierce, Rockford, IL, USA). Samples containing equal amounts of protein were mixed with 2x Laemmli sample buffer and incubated at 95 °C for 3 min, followed by separation on 9 or 12 % polyacrylamide gels and transfer to polyvinylidene difluoride (PVDF) membranes. Blots were blocked in 5 % non-fat dried milk in phosphate-buffer saline (PBS) containing 0.05 % Tween-20, and probed with primary antibodies, followed by secondary peroxidase-labeled anti-rabbit or mouse IgG. The Can Get Signal Immunoreaction Enhancer Solution (Toyobo, Osaka, Japan) was occasionally incubated with primary antibodies to enhance immunoreaction. Protein expression was detected with a chemiluminescence reagent (Perkin-Elmer, Boston, MA, USA), and the resulting images examined with a LAS-1000 (Fuji Film, Tokyo, Japan) image analyzer. β-Actin was used as the internal control to normalize the levels of proteins of interest.
4
Immunoblot Analysis of Metabolic Enzymes
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Immunoblot analysis was performed according to standard protocols, as previously described [31 (link)]. In brief, cells were washed with PBS and lysed in radioimmunoprecipitation buffer. The resulting lysates were fractionated by SDS-polyacrylamide gel electrophoresis, and the separated proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA) and exposed to primary antibodies to MCT2 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-166925), MCT4 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-376140), LDHA (Thermo Fisher Scientific, Rockford, IL, USA, PA5-27406), LDHB (Abcam, Cambridge, UK, ab85319), and to α-tubulin (Santa Cruz Biotechnology, Dallas, TX, USA, sc-32293). Immune complexes were detected with HRP-conjugated secondary antibodies and Chemiluminescence Reagent (PerkinElmer, Waltham, MA, USA).
5
Immunoblotting Analysis of Retinal Proteins
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Retinae were isolated and homogenized in 0.1% SDS lysis buffer, and protein concentration was determined by Bradford assay (Bio-Rad Laboratories, München, Germany). Proteins were subsequently separated in a 4–20% gradient TGX Gel and immunoblotted to a polyvinylidene difluoride membrane using a semi-dry turboblot system (all from Bio-Rad, München, Germany). After blocking unspecific binding using 5% nonfat dry milk in TBS, containing 0.1% Tween (Sigma-Aldrich, Darmstadt, Germany), the membranes were sequentially incubated with the primary antibodies against pErk1/2, Erk1/2, pAkt, and Akt (all from Cell Signaling Technology, Frankfurt/Main, Germany) at 4°C overnight. After the washing steps, a horseradish peroxidase-conjugated swine anti-rabbit antibody was used for immunodetection (DakoCytomation, Hamburg, Germany). Immunoreactive bands were visualized by incubation in a chemiluminescence reagent (PerkinElmer, Boston, MA, United States), and signals were detected with the Fusion SL (VWR, Darmstadt, Germany). Membranes were stripped with a reprobing buffer containing 20% sodium dodecyl sulfate between immunodetection and incubation with subsequent primary antibodies. Integrated densities were measured with the ImageJ software (Abràmoff et al., 2004 ).
6
Western Blot Analysis of Cell Signaling
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Cells were lysed using lysis buffer containing 150 mM NaCl, 1% NP40, 0.5% deoxycholic acid, 0.1% SDS, 50 mM Tris (pH 8.0), and a 1:25 protease inhibitor cocktail for total protein. The protein concentration of the lysates was detected by the Bradford protein assay system (BCA; Bio-Rad). Protein samples were subject to SDS-PAGE, and transferred onto PVDF membranes. Membranes were blocked and incubated with primary antibodies at 4°C overnight. Primary antibodies, including anti-NOTCH1, anti-LDHA1, anti-E-cadherin, anti-N-cadherin, anti-Snail1, anti-TGF-β, anti-Smad3, anti-p-Smad3 were from Abcam (Cambridge, UK), and anti-actin and anti-GAPDH were from Santa Cruz Biotech (Santa Cruz, USA), and were used at a 1:1000 dilution. Then, membranes were incubated with goat anti-rabbit/mouse IgG (H+L)-HRP (Santa Cruz Biotech) secondary antibody at room temperature for 2 h. The protein signals were visualized using chemiluminescence reagent (PerkinElmer, Waltham, USA) and detected with an enhanced chemiluminescence western blotting detection system (Amersham Bioscience, London, UK).
7
Extraction and Western Blot Analysis of Intestinal Tumor Proteins
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Mouse intestinal tumors were homogenized on ice in CelLytic MT (Sigma‐Aldrich) supplemented with protease inhibitor cocktail (Sigma‐Aldrich), 100 mmol/L NaF, and 1 mmol/L Na3VO4. The extracts were centrifuged at 20 000 g for 15 minutes at 4°C, and the resulting supernatants were used for experiments. Cellular proteins were extracted with CelLytic M (Sigma‐Aldrich) supplemented with protease inhibitor cocktail, 100 mmol/L NaF, and 1 mmol/L Na3VO4, centrifuged 15 000 g for 15 minutes, and the supernatants were collected. Equal amounts of total proteins were separated by SDS‐PAGE under reducing conditions using 4%‐12% gradient gels and transferred onto an Immobilon‐P membrane (Millipore). After blocking with 5% nonfat milk in 25 mmol/L TBS with 0.1% Tween‐20, pH 7.6 (TBS‐T), the membranes were incubated overnight at 4°C with primary Ab, followed by washing with TBS‐T and incubation with HRP‐conjugated secondary Ab diluted in TBS‐T with 1% BSA for 1 hour at room temperature. The labeled proteins were visualized with a chemiluminescence reagent (PerkinElmer Life Science). The following primary Abs were used: anti‐β‐actin mouse mAb (Sigma‐Aldrich), anti‐PAR‐2 rabbit mAb, anti‐phospho‐NF‐κB p65 (Ser536) rabbit mAb and anti‐NF‐κB p65 rabbit mAb (Cell Signaling Technology Japan).
8
Western Blot Analysis of Osteoclast and Osteoblast Markers
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The cells designated for protein extraction from in vitro osteoclastogenesis and osteoblast differentiation assays were directly lysed in the tissue culture plates at different time points using RIPA Cell Lysis Buffer. Western blotting was performed as described previously33 (link). Antibodies used were as follows: Anti-mouse Morc3 (MBL International. Japan); Anti-mouse NFATc1 (BD Biosciences, USA); Anti-mouse V-ATPase d2 subunit (Produced for the Centre for Orthopaedic Research, UWA38 (link)); Anti-mouse DC-STAMP (Merck Millipore, Germany); Anti-rabbit c-FOS (Cell Signaling Technology, USA); Anti-rabbit Phospho-STAT1 (Tyr 701) (Cell Signaling Technology, USA); Anti-rabbit STAT1 (Cell Signaling Technology, USA); Anti-rabbit Rankl (R&D systems, China); Anti-goat OPG (R&D systems, China); Anti-rabbit β-catenin (Cell Signaling Technology, USA) and Anti-mouse β-Actin (JLA-20) (Developmental Studies Hybridoma Bank. USA). Detection was done by respective peroxidase-conjugated antibodies (Sigma-Aldrich, USA) and chemiluminescence reagent (PerkinElmer Life Sciences).
9
Frataxin Protein Detection Protocols
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Four protein standards were used in this study. Cynomolgus monkey mature frataxin (aa 81–210) was purchased from LifeSpan BioSciences (LS-G21788). Mouse recombinant intermediate form of frataxin (aa 41–207) was obtained from LifeSpan BioSciences (LS-G14956). Human and mouse mature frataxin were prepared using a protocol described previously (Guo et al. Anal. Chem.). Five antibodies were tested here, including AB113691, AB175402, AB124680 obtained from Abcam (Cambridge, MA), MAB1594 from Millipore-Sigma (Billerica, MA), and LS-C197243 from LifeSpan BioSciences (Seattle, WA). Anti-rabbit HRP and anti-mouse HRP were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX). Stainless steel beads for tissue homogenization were purchased from Next Advance (Troy, NY). NuPAGE™ LDS sample buffer was from Thermo Scientific (Waltham, MA) and chemiluminescence reagent (NEL103E001) was purchased from Perkin Elmer (Waltham, WA). Costar® Spin-X centrifuge tube filters (0.22 μm cellulose acetate) were obtained from Corning (Corning, NY).
10
Western Blot Analysis of Protein Expression
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30 μg of protein from whole cell lysates was resolved on 10% SDS-PAGE gels (Bio-Rad Laboratories, Hercules, CA) and transferred onto PVDF membranes (GE Healthcare, Piscataway, NJ). After transfer, membranes were blocked in 5% nonfat milk in TBST and membranes were incubated with specific antibodies (overnight at 4°C) followed by horseradish peroxidaseconjugated anti-mouse or anti-rabbit (Santa Cruz, Dallas, TX) immunoglobulin for 1 hour at RT. Signal was detected by Chemiluminescence Reagent (PerkinElmer, Waltham, MA) and visualized by autoradiography. Anti-human survivin antibody was purchased from R&D Systems (Minneapolis, MN). Anti-CUG-BP1, anti-HuR, and anti-GAPDH antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). All primary anti-bodies were used at a dilution of 1:2000 and all secondary antibodies were used at a dilution of 1:4000. Signal intensity was quantified using Image Lab quantification software (Bio-Rad, Hercules, CA).
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