This method was performed as described previously17 (link). The cells grown on plates were trypsinized, and detached cells were collected by centrifugation. The cell pellet was lysed with cold lysis buffer supplemented with protease inhibitors. Protein samples (30 µg) from each cell lysate were equivalently loaded on a precast gel and used for electrophoresis. Gels were subsequently blotted onto nitrocellulose membranes (0.45 μM; Bio-Rad, Hercules, CA), followed by blocking nonspecific binding with a solution containing 1 × PBS, 0.1% Tween-20, and 5% nonfat dry milk powder at room temperature for 1 h. Membranes were incubated with the primary antibodies anti-E-cadherin, anti-Snail, anti-Vimentin, anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA) and anti-Sema4C (Abcam, Cambridge, MA, USA) at 4 °C overnight. After extensive washing with TBST, horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad) were incubated for 1 h at room temperature. Bands were detected with an enhanced chemiluminescence kit (Super Signal West Pico Substrate; Pierce, Rockford, IL, USA). Quantification of signal intensities was performed by densitometry on a Xerox scanner using NIH ImageJ software (ImageJ, Bethesda, MD).
Horseradish peroxidase hrp conjugated secondary antibody
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
Horseradish peroxidase (HRP)-conjugated secondary antibodies are a type of laboratory reagent commonly used in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry. These antibodies are designed to bind to the primary antibody that is specific to the target antigen, and the attached HRP enzyme can then be used to catalyze a colorimetric or chemiluminescent reaction, allowing for the detection and visualization of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (9 mentions)
β actin, by Merck Group (7 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Enhanced chemiluminescence reagent, by Bio-Rad (4 mentions)
Lysis buffer, by Merck Group (4 mentions)
Anti pparγ, by Santa Cruz Biotechnology (4 mentions)
Anti phospho fak, by Cell Signaling Technology (4 mentions)
Anti phospho akt, by Cell Signaling Technology (4 mentions)
Anti gapdh, by AbFrontier (4 mentions)
Anti phospho pi3k p110α, by Cell Signaling Technology (4 mentions)
Anti phospho mtor, by Abcam (4 mentions)
Anti leptin, by R&D Systems (4 mentions)
Anti tnf α, by Santa Cruz Biotechnology (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Vimentin, by Cell Signaling Technology (4 mentions)
Chemidoc mp imaging system, by Bio-Rad (4 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Image lab software, by Bio-Rad (3 mentions)
Trans blot turbo transfer system, by Bio-Rad (3 mentions)
77 protocols using horseradish peroxidase hrp conjugated secondary antibody
1
Western Blot Analysis of EMT Markers
2
Tau and Autophagy Protein Antibodies
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Anti–pThr-212/pSer-214/pThr-217 tau AT100 (MN1060) and anti-human tau HT7 (MN1000) were from Thermo Fisher; anti-GAPDH (MAB374) was from EMD Millipore; anti-Rab5 (ab18211), anti-p62 (ab91526), anti-NDP52 (ab68588), anti–galectin-8 (ab183637), and anti-ubiquitin (ab7780) were from Abcam; anti-p62 (ab610832) was from BD Biosciences; anti-LC3 (NB1000–2220) and anti-NDP52 (BO01P) were from Novus Biologicals; anti-optineurin (100000) was from Cayman; anti-FIP200 (17250–1-AP) was from Proteintech Group; anti-ubiquitin P4D1 (sc-8017) was from Santa Cruz Biotechnology; and anti–galectin-8 (AF1305) was from R&D Systems. Horseradish peroxidase (HRP)–conjugated secondary antibodies were from Bio-Rad. Alexa Fluor–conjugated secondary antibodies were from Life Sciences.
3
Immunocytochemistry and Western Blot Antibodies
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The following primary antibodies were used for immunocytochemistry and Western blot analysis: rabbit polyclonal antibodies against lamin B1, Tpr, importin α7 (Abcam, Cambridge, MA), pHIST3 (serine 10), MAP-2 (Merck Millipore, Darmstadt, Germany), actin, βIII-tubulin (Sigma-Aldrich, Milano, Italy), pERK, pCREB (Cell Signaling, Beverly, MA), lamin A/C (Santa Cruz Biotechnology, Dallas, TX), synaptophysin (SySy, Gottingen, Germany), and drebrin (Enzo Life Sciences, Farmingdale, NY); and mouse monoclonal antibodies against Nup153, lamin B2, CREB (Abcam), nuclear pore complex (mAb414; Covance, Princeton, NJ), Lap2β (BD Biosciences, East Rutherford, NJ), Tau (Tau-1; Merck Millipore), ERK (Cell Signaling), importin β (Sigma-Aldrich), and Alexa 488–conjugated Tuj1 (Covance). For Western blot analysis, horseradish peroxidase (HRP)–conjugated secondary antibodies (Bio-Rad, Hercules, CA) were used for detection. For immunofluorescence analyses, Alexa fluorophore–conjugated anti-mouse, anti-rabbit, and anti-rat antibodies from Invitrogen (Thermo Fisher Scientific, Waltham, MA) were used. Unless otherwise specified, general reagents and chemicals were from Sigma-Aldrich, and reagents for cell cultures were from Invitrogen.
4
Protein Blotting Antibody Validation
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Antibodies used for protein blotting were TBL1XR1 (Novus), HDAC3 and NCoR (Abcam) and alpha-tubulin (Santa Cruz). Horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad) were used to detect primary antibodies.
5
Western Blot Analysis of Chondrocyte Differentiation
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Western blot (WB) analyses were performed using 3D pellet samples at day 17 and day 24 of differentiation. Briefly, cells were lysed in lysis buffer (150 mM NaCl, 1% NP-40, 0.5% Deoxycholic Acid, 0.1% SDS, 50 mM Tris pH 8.0) containing protease inhibitor cocktails (Sigma, St. Louis, MO). Proteins of lysate supernatant were separated with SDS-polyacrylamide gel electrophoresis, transferred to PVDF membrane (Bio-Rad, Hercules, CA) and immunoblotted with primary antibodies of collagen II, aggrecan, and HSP70, followed by incubation with the horseradish peroxidase (HRP) conjugated secondary antibodies. All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Tetramethylbenzidine (TMB) substrate kit (Vector Laboratories, Burlingame, CA) was used to visualize the protein bands. Membranes were dried and scanned into digital images. Protein bands were analyzed quantitatively using Image Pro Plus software (Media Cybernetics). The protein expression levels were presented as ratios of total intensity of the protein bands normalized by that of the actin band from the same sample on the same membrane to minimize the protein loading variation.
6
Western Blot Analysis of ADAM10 Protein
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Total protein from cells and tissues were extracted with RIPA buffer containing protease inhibitor cocktail and EDTA. Proteins were electrophoresed and transferred to membranes as described previously36 (link). The membranes were then blocked with 5% Blotting-Grade Blocker (Bio-Rad) in TBS-T (50 mM Tris, 138 mM NaCl, pH 7.4, 0.1% Tween-20) and subsequently incubated at 4 °C overnight with rabbit anti-ADAM10 polyclonal antibody (LifeSpan BioSciences,) or rabbit anti-α-tubulin polyclonal antibody (Cell Signaling Technology) followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad).
7
Antibody Specificity and Sources
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Antibodies against Tap and the human THO/TREX components have been previously described (47 (link),48 (link)). Rabbit polyclonal antibodies against GFP, mouse IgG, CBP20, SLBP, and SARS-CoV-2 spike (S) protein, mouse monoclonal antibodies against ALYREF, SC35, β-actin, FLAG-peptide tag, Rho-1D4 tag, Strep-tag II and a rat monoclonal antibody against U1C were commercially acquired. Antibodies against SARS-CoV-2 Nsp14 and puromycin were obtained from MRC PPU Reagents and Services, University of Dundee and DSHB, University of Iowa, respectively. Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Bio-Rad, whereas Alexa- and DyLight-conjugated secondary antibodies were purchased from Thermo Fisher Scientific. HRP-conjugated anti-rat IgG was purchased from Abcam. The monoclonal antibody 38A1 against CBP80 (49 (link)) was a gift from Drs Hito Ohno and Ichiro Taniguchi of Kyoto University. Details of the primary antibodies used in this study are listed in Supplementary Table S1 .
8
Western Blot Analysis of Hypoxia Markers
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Liver fragments were lysed in Protein Solubilization Buffer (PSB, Bio-Rad, USA) with Complete Protease Inhibitor Cocktail (Roche, USA), homogenized with pestle and centrifuged 30 min at 14,000 g. Thereafter, 2x loading buffer was added to the supernatant, and the sample was incubated at 65 °C for 5 min. For the protein separation, 10%–12.5% SDS-PAGE was performed. Transfer from the gel to PVDF membranes by semi-wet approach was conducted using Trans-Blot® Turbo™ RTA Mini LF PVDF Transfer Kit (Bio-Rad, USA). The membranes were blocked with milk (5%) in Tris-buffered saline with Tween 0.1% (TTBS) for 1 h at room temperature, then incubated overnight with primary antibodies to HIF1α (ab179483, 1:1000, abcam) and GAPDH (sc-25778, 1:1000, Santa Cruz), PHD2 (ab244389, 1:500, abcam) overnight at 4 °C with gentle shaking. Thereafter samples were stained with horseradish peroxidase (HRP) conjugated secondary antibodies (Bio-Rad, USA) for 1 h at room temperature. Target proteins were visualized by Novex ECL Kit (Invitrogen™ Thermo Fisher Scientific, USA) in ChemiDoc (Bio-Rad, USA). For optical density measurements of the protein bands Image Lab Software tool was used with GAPDH as a reference protein.
9
Autophagy and Apoptosis Signaling Assays
10
Western Blotting for Protein Analysis
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In brief, the protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and further transferred onto PVDF membranes (Millipore Corp.). After blocking in Tris-buffered saline (TBS, pH 7.6) containing 5% non-fat milk and 0.1% Tween-20, the membranes were probed with primary antibodies at 4°C overnight. After washed with TBST (TBS and 0.1% Tween-20), the membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad). Proteins were further detected by the SuperSignal West Pico Chemiluminescent Substrate kit (Pierce). The image was scanned with GS800 Densitometer Scanner (Bio-Rad), and processed with PD Quest 7.2.0 software (Bio-Rad).
The above-mentioned primary antibodies included rabbit anti-MAG (1:800), chickenanti-P0 (1:800), mouse anti-β-actin (1:4000), rabbit anti-p-AKT (1:1000), mouse anti-AKT (1:1000), rabbit anti-ERK1/2 (1:1000), rabbit anti-p-ERK1/2 (1:1000).
The above-mentioned primary antibodies included rabbit anti-MAG (1:800), chickenanti-P0 (1:800), mouse anti-β-actin (1:4000), rabbit anti-p-AKT (1:1000), mouse anti-AKT (1:1000), rabbit anti-ERK1/2 (1:1000), rabbit anti-p-ERK1/2 (1:1000).
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