For CCS (copper chaperone to superoxide dismutase) Western blot, cells were grown on six-well plates, transfected and treated with the specified compounds. Cells were washed once with ice-cold 1× PBS. Lysis buffer [20 mM Hepes, pH 7.4, 75 mM NaCl, 1.5 mM MgCl2, 2 mM EGTA, 2 mM DTT and 0.5% (v/v) Triton-X100], supplemented with protease and phosphatase inhibitors, was added to each well and cells were incubated for 1 h at 4°C on a shaker. Cells were scraped, transferred to a tube and centrifuged at 16000 g for 10 min at 4°C. Supernatant was collected and equal amounts of protein per condition were incubated at 100°C for 5 min in 2× Laemmeli sample buffer (BioRad). Samples were loaded on a 12% TGX polyacrylamide gel (BioRad), run at 250 V for 40 min and transferred to PVDF membrane (Millipore). Rabbit anti-CCS antibody was a kind gift from Dr Dennis Thiele. HRP-conjugated anti-rabbit secondary antibody was incubated for 1 h at room temperature. Immunodetection was performed with the Luminata Forte HRP substrate (Millipore). Band densities were measured using ImageJ (NIH). For LC3 detection, rabbit anti-LC3 antibody was used. HRP-conjugated anti-rabbit secondary antibody was incubated for 1 h at room temperature. Immunodetection was performed with the Luminata Forte HRP substrate (Millipore). Band densities were measured using ImageJ.
Luminata forte hrp substrate
Manufactured by Merck Group
Sourced in United States
Luminata Forte HRP substrate is a chemiluminescent substrate used for the detection of horseradish peroxidase (HRP) in western blotting and other immunoassay applications. The substrate generates a luminescent signal upon reaction with HRP, which can be detected and quantified using a luminometer or imaging system.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Protran, by Cytiva (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Whatman, by Cytiva (3 mentions)
Hyperfilm, by GE Healthcare (3 mentions)
Anti mouse igg hrp, by GE Healthcare (2 mentions)
Las 4000 mini, by GE Healthcare (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Peroxidase labeled secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Erk1 2, by Cell Signaling Technology (2 mentions)
α tubulin, by Cell Signaling Technology (2 mentions)
Phospho p38, by Cell Signaling Technology (2 mentions)
Phospho jnk, by Cell Signaling Technology (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Phospho erk1 2, by Cell Signaling Technology (2 mentions)
Snail, by Cell Signaling Technology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Chemidoc mp system, by Bio-Rad (2 mentions)
Nitrocellulose electro blotted membranes, by Cytiva (2 mentions)
39 protocols using luminata forte hrp substrate
1
CCS and LC3 Western Blot Protocol
2
Western Blot Analysis of Protein Expression
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Cells were harvested and lysed in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS/PAGE) sample buffer (50 mm Tris/HCl, pH 6.8, 2% SDS, and 10% glycerol). Protein concentration was determined by Bio‐Rad DC protein assay (Bio‐Rad). Proteins were separated by SDS/PAGE and transferred onto a polyvinylidene fluoride membrane (Millipore, Carrigtwohill, Ireland). The membrane was blocked with 1% skimmed milk in Tris‐buffered saline containing 0.05% Tween 20 and probed with appropriate antibodies using the Envision system (Dako, Glostrup, Denmark). Signals were developed by Luminata Forte HRP substrate (Millipore) or SuperSignal West Femto Substrate (Thermo Fisher Scientific, Rockford, IL, USA) and then captured by ChemiDoc MP Imaging System (Bio‐Rad). Following primary antibodies were used: anti‐p53 antibody (FL393; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti‐p21 antibody (H164; Santa Cruz Biotechnology), anti‐CES2 (G5; Santa Cruz Biotechnology), and anti‐GAPDH antibody (6C5; Santa Cruz Biotechnology).
3
Western Blot Analysis of CCL5-Stimulated hBMECs
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Western blotting was performed as previously described (93 (link)). hBMECs were starved overnight and collected or stimulated with 100 ng/ml CCL5 for 10 min before harvest. Cells were washed with phosphate-buffered saline (PBS) and lysed in 1% NP-40 buffer with a protease inhibitor cocktail (Sigma) as previously described (12 (link)). Total protein levels were determined in a bicinchoninic acid assay (Thermo Fisher), and proteins were resolved by SDS–12% PAGE, transferred to nitrocellulose, blocked in PBS–1% bovine serum albumin (BSA), and incubated with antibodies in a blocker. Proteins were detected using horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies (Amersham) and the Luminata Forte HRP substrate (Millipore).
4
Developmental Regulation of Myelin Proteins
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CC and cortices from P7, P15 and P30 WT and JNK1KO mice, after brain sectioning with Leica vibratome, were obtained by dissection. Tissue lysates were obtained adding RIPA buffer (1% NP40, 150 mM NaCl, 50 mM TRIS HCl pH 8, 5 mM EDTA, 0.01% SDS, 0.005% Sodium deoxycholate, Roche protease inhibitors, PMSF) for 10 min at 4 °C. Samples were homogenized on ice with a pellet pestle (Sigma-Aldrich, Saint Louis, MS, USA) and centrifuged at 1300 rpm at 4 °C. For immunoblots, equal amounts of proteins were resolved by SDS–PAGE and blotted to nitrocellulose membranes, which were then probed with anti-MBP (1:1000, Millipore, Billerica, MS, USA—MW: 18–21 kDa), -CNPase (1:500, Sigma-Aldrich, Saint Louis, MS, USA—MW: 47 kDa), -MOG (1:1000, Proteintech, Manchester, UK—MW: 25 kDa) and -SMI31 (1:1000, SMI-31R Sternberger—MW: 160–200 kDa) antibodies. The membranes were subsequently incubated with the secondary antibodies and developed using the Luminata Forte HRP substrate (Millipore, Billerica, MS, USA). Signals are normalized using anti-β-Tubulin (1:5000, Sigma-Aldrich, Saint Louis, MS, USA—MW: 50 kDa) and anti-Vinculin (1:2000, Sigma-Aldrich, Saint Louis, MS, USA—MW: ~ 120 kDa) antibodies. Blots were imaged on a ChemiDoc (Bio-Rad) and analyzed using Image Lab software.
5
Detecting OsWRKY42 Protein in Rice Leaves
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Leaves of fourteen days old TN-1 rice seedlings were infiltrated with saturated Agrobacterial cultures of LBA4404/ pMDC7::OsWRKY42 with or without estradiol. After 16–18 h, the infiltrated region was cut and ground in liquid nitrogen followed by homogenization in lysis buffer (50 mM Tris-HCl [pH 7.5], NaCl [150 mM], Mannitol [250 mM], EDTA [5 mM], 10% Glycerol, 1 mM DTT 1% TritonX100, 1 mM PMSF, 1 mM NaF) with plant protease inhibitor cocktail (Sigma-Aldrich) [42 (link)]. Total protein supernatants were isolated after centrifugation at 15000 g for 15 min at 4 °C to remove cellular debris. Equal amounts of isolated protein supernatants were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The OsWRKY42 protein was detected by Western blot analysis using anti-FLAG (AbCAM) antibodies. HRP conjugated anti-rabbit secondary antibody (AbCAM) was probed and the protein band was viewed using Luminata Forte HRP substrate (Millipore). The signal was captured under Vilber Lourmat chemiluminescence imaging system with Chemi-capt 5000 software (version 12.8; Vilber Lourmat).
6
Whole Cell Extract Preparation and Western Blotting
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Cells were harvested and washed using STOP buffer (150 mM NaCl, 50 mM NaF, 10 mM EDTA, 1 mM NaN3 (pH 8.0)). Cell pellet was mixed with equal amount of buffer 1 (50 mM Tris-HCl (pH 7.5), 1.5 mM MgCl2, 300 mM NaCl, 0.3% NP-40, 1 mM DTT, cOmplete™ Protease Inhibitor Cocktail (Sigma), PhosSTOP™ (Sigma)), and the mixture was beaten with glass beads using Fastprep bead-beater. Buffer 2 (50 mM Tris-HCl (pH 7.5), 1.5 mM MgCl2, 7.5% glycerol, 1mM DTT, cOmplete™ Protease Inhibitor Cocktail, PhosSTOP™) was added in volumes 2x the cell pellet, and the mixture was centrifuged (5k rpm, 2 min once, and 14k rpm, 10 min twice) to obtain supernatants (whole cell extracts). Whole cell extracts were separated by SDS-PAGE using 5–20% gels, and transferred to PVDF membranes (Immobilon P, Millipore). 5% skim milk in TBST was used for blocking. The following antibodies were used. Primary antibodies: anti-FLAG (Sigma, F1804, 1:1,000); anti-tubulin (TAT133 (link)). Secondary antibodies: anti-mouse IgG-HRP (GE, NA931V, 1:10,000). Chemiluminescence was generated by Luminata Forte HRP substrate (Millipore) and detected by LAS 4000 mini (GE).
7
Western Blot Protein Extraction and Detection
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Protein extracts were obtained by lysis in ice‐cold RIPA buffer for 30 min followed by 30‐min centrifugation at 4°C. Proteins were separated on 4–20% acrylamide gradient SDS gels (Bio‐Rad) and transferred to Amersham Protran 0.45‐μm nitrocellulose membranes (GE Healthcare Life Sciences) for 90 min at 300 mA. Further, the membranes were blocked in 5% skim milk in PBST buffer and incubated thereafter with the primary and secondary antibodies. Membranes were developed using the Luminata Forte HRP substrate (Millipore) and exposed in the Fusion Solo apparatus (Vilber Lourmat).
8
Organotypic SCN Slice Culture and Analysis
9
Immunoblot Analysis of DH82 Cell Proteins
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Proteins of DH82 cells were extracted using Ez RIPA buffer (Atto, Japan) and quantitated it using Bradford assay (Bio-Rad Laboratories, USA). Next, 10 µg of the whole lysate in each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Amersham, GE Healthcare, USA). Blocking buffer (5% skim milk in PBS with 0.1% Tween 20) was added for 1 hour at room temperature. Primary antibodies, including total-STAT3 (Cell Signaling, USA), p-STAT3 (Cell Signaling), JAK2 (Cell Signaling), inducible nitric oxide synthase (iNOS; Santa Cruz Biotechnology, USA), cyclooxygenase-2 (COX2; Cell Signaling), nuclear factor kappa B (NF-κB; Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology), were added in a dilution of 1:1000 and incubated for 16 hours at 4 °C. Then secondary anti-rabbit or anti-mouse antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology) was added, and bands were detected using the Luminata Forte HRP substrate (Millipore, USA).
10
Quantifying 5hmC and 5mC Levels
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Total genomic DNA was extracted using the DNeasy Kit (QIAGEN), denatured (0.4M sodium hydroxide, 10mM EDTA at 100°C for 10 min), and then neutralized (6.6M cold ammonium acetate, pH 7). We applied 200 ng of DNA to a prewet Amersham Hybond‐N+ membrane (GE Healthcare Life Sciences). The membrane was blocked and incubated in primary antibody overnight (anti‐5hmC and anti‐5mC 1/200; Active Motif, Inc., Carlsbad, CA, USA), followed by the appropriate peroxidase‐labeled secondary antibody (1/5000; Santa Cruz Biotechnology). Blots were visualized using Luminata Forte HRP substrate (Millipore), and quantified using ImageJ software (NIH; https://imagej.nih.gov/ij/).
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