Protein extraction from the sample and concentration determination of whole cells was performed as previously described (Duan et al., 2016 (link)). Briefly, the cells were lyzed in RIPA buffer containing phenylmethanesulfonyl fluoride (PMSF) and protease inhibitor cocktail. Equal amounts of proteins were resolved on SDS polyacrylamide gels and transferred to nitrocellulose membrane. The resulting blots were blocked with 5% non-fat dry milk and probed with antibodies. The following antibodies were used: GAPDH (KangChen, Shanghai, China), β-actin (Santa Cruz, Santa Cruz, CA, United States), MICAL1 (proteintech, Hubei, China), NEDD9 (Santa Cruz), FLAG (Abways, Shanghai, China), Rac1 (BD, Franklin Lakes, NJ, United States), RhoA, Cdc42, and HA antibodies (Cell Signaling, Danvers, MA, United States). Protein bands were detected by incubating with HRP-conjugated antibodies (Santa Cruz) and developed using ECL reagent (Millipore). Digital images of the positive bands were obtained and analyzed with Quantity One (Bio-Rad, Hercules, CA, United States).
Hrp conjugated antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
HRP-conjugated antibodies are a type of antibody that have been chemically conjugated to horseradish peroxidase (HRP), an enzyme commonly used in various immunoassays and detection techniques. The HRP-conjugated antibodies can be used to detect and quantify target proteins or other biomolecules in a wide range of applications, such as Western blotting, ELISA, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Pierce ecl, by Thermo Fisher Scientific (6 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Ecl reagent, by Merck Group (2 mentions)
Ponceau s, by Merck Group (2 mentions)
Tween 20, by Merck Group (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Mab2259, by Merck Group (2 mentions)
Mab1622, by Merck Group (2 mentions)
E cadherin, by Santa Cruz Biotechnology (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Anti β catenin, by Cell Signaling Technology (2 mentions)
Anti gsk3β, by Cell Signaling Technology (2 mentions)
Anti gapdh antibody, by GeneTex (2 mentions)
Anti wnt3a, by R&D Systems (2 mentions)
31 protocols using hrp conjugated antibody
1
Protein Extraction and Western Blot Analysis
2
Extracellular Vesicle Isolation and Characterization
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In total, 200 ml of cell culture supernatant was collected followed by centrifugation to remove intact cells (500 g for 5 min) and cellular debris (20,000 g for 15 min). EVPs were isolated by ultracentrifugation following MISEV2018 EVP isolation protocol. The EVP pellets were resuspended in 30 µl of PBS and cryopreserved at -80°C. EVP concentration and size distribution were determined with Nanosight NS300. Five microliters of EVP samples was placed on 300 mesh grids and incubated for 3 min at room temperature (RT). Excess samples were blotted with filter paper and stained with 1% uranyl acetate for 5 min. Samples were then examined under TEM (Hitachi). The Western blot was routinely performed. After lysis with RIPA buffer, Mini-PROTEAN Tetra Handcast System (BioRad) and Trans-Blot Turbo Transfer System (BioRad) were used for electrophoresis and subsequent transferring. The protein blot was blocked for 1 h with 5% skimmed milk in PBS/0.05% Tween 20 and incubated for 6 h at 4°C with Santa Cruz Biotechnology HRP conjugated antibodies against TSG-101 (sc-7964, 1:500), CD81 (sc-166029,1:500), CD63 (sc-100304, 1:500), and GAPDH (sc-47724, 1:1000). Samples were washed with PBS/0.05% Tween for 10 min 3 times. Blots were developed with chemiluminescence (BioRad).
3
Western Blot Analysis of NOTCH1 Signaling
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Cells were lysed in RIPA buffer (Sigma) and protein concentration was estimated using BCA (MP biomedicals) method [59 (link)]. Forty microgram protein was separated on 10% SDS-PAGE gel, transferred to nitrocellulose membrane and transfer was verified using Ponceau S (Sigma). Later the blots were blocked in Tris-buffered saline containing 5% BSA (Sigma) and 0.01% Tween-20(Sigma) and were probed with full-length NOTCH1 (sc-6014-R, Santacruz biotechnology), anti-activated NOTCH1 antibody (ABCAm; ab8925) and anti-actin (A5316, Sigma) antibody. The membranes were then incubated with corresponding secondary HRP-conjugated antibodies (Santa Cruz Biotechnology, USA) and the immune complexes were visualized by Pierce ECL (Thermo Scientific, USA) according to manufacturer's protocol. Western blot experiments were performed in triplicate.
4
Immunoblotting of proteasome subunits
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EndoC-βH1 cells were lysed in RIPA buffer supplemented with a protease inhibitor cocktail (Roche Applied Science, Germany). Protein quantification was performed using the BCA protein assay kit (Thermo Fisher Scientific, USA). A total of 25 µg of protein was subjected to electrophoresis on 12% acrylamide/bis acrylamide SDS page gels. After electrophoresis, proteins were transferred onto nitrocellulose membranes (GE Healthcare, USA). Membranes were stained with primary antibodies overnight at 4°C and secondary HRP-conjugated antibodies (Santa Cruz Biotechnology, USA) for 1h at room temperature. Primary antibodies were from Enzo Life Sciences (Switzerland) and were used at a dilution of 1:1000 (β1i: BML-PW8345; β5i: BML-PW8355; β2i: BML-PW8350). The loading control was β-actin (MAB1501, EMD Millipore, USA) and was used at 1:1000 dilution. Secondary antibodies were anti-mouse (#G21040) or anti-rabbit (#sc-2004) antibodies from Santa Cruz Biotechnology and were used at a dilution of 1:5000. Western ECL substrate was used for imaging (1705062, BioRad, USA).
5
Western Blot Analysis of Protein Markers
6
Protein Extraction and Analysis of ALS Mouse Tissues
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The lungs and spleens of melittin- or saline-treated hSOD1G93A transgenic mice were dissected and homogenized in RIPA buffer (50 mM Tris-Cl pH 7.4, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), and 150 mM NaCl) containing a protease inhibitor cocktail (Calbiochem, CA, USA). Homogenized tissues were centrifuged at 14,000 rpm for 20 min at 4℃. Total protein was quantified using the BCA assay kit (Pierce, IL, USA). Samples denatured with SDS sampling buffer were separated through SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane for Western blot. For the detection of target proteins, the membranes were blocked with 5% non-fat milk in tris-buffered saline (TBS) and then incubated with various primary antibodies: anti-tubulin (Abcam, Cambridge, UK), anti-phospho-ERK (Cell Signaling Technologies), anti-Bcl2 (SantaCruz, CA), anti-HO1 (Abcam, Cambridge, UK), anti-Iba-1 (Wako, Osaka, Japan), anti-CD14 (BD Pharmingen), anti-COX2 (BD Biosciences), and anti-TNF-α (Abcam, Cambridge, UK). The blots were then probed with HRP-conjugated antibodies (Santa Cruz Biotechnology, CA, USA) and visualized by using enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia, NJ, USA). An LAS-3000 image analyzer was used for detecting immunoblotted bands (Fujifilm, Tokyo, Japan).
7
Western Blotting for Inflammation Mediators
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Western blotting was carried out as previously described (Newson et al., 2014 (link)). Briefly, cells from peritoneal washouts or ex vivo culture were lysed in RIPA buffer with protease inhibitors (both Sigma) and the protein concentration determined by Bradford assay (Bio-Rad). Ten micrograms of protein were separated by SDS-PAGE (National Diagnostics). Separated proteins were transferred onto a polyvinylidene fluoride membrane (Immobilon; Millipore) and incubated with COX-1, COX-2, mPGES-1, mPGE-2, EP1-4 (Cayman Chemical), β-actin, and GAPDH (Sigma) overnight in block buffer (Tris-HCL, 1% Tween-20, 1% BSA [Sigma], and 5% nonfat milk [Marvel]). Blots were washed and incubated with HRP-conjugated antibodies (Santa Cruz Biotechnology) for 1 hr at room temperature in blocking buffer. Specific proteins were visualized by enhanced chemiluminescence (ECL) hyperfilm.
8
Western Blot Analysis of NRBP1 and ERK1/2
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Cells were lysed in RIPA buffer and protein concentration was estimated using BCA method [43 (link)]. 50 and 100 μg protein was used for NIH-3 T3 and HNSCC cell lines western analysis. The protein was separated on 10 % SDS-PAGE gel, transfer was verified using Ponceau S (Sigma), transferred on nitrocellulose membrane and blocked in Tris-buffered saline containing 5 % BSA (Sigma) and 0.05 % Tween-20(Sigma). Later, blots were probed with anti-NRBP1 (Santa Cruz Biotechnology; sc-390087), anti-total ERK1/2 (Cell signaling; 4372S), anti-Phospho ERK1/2 (Cell signaling; 4370S) and anti- GAPDH antibody (Santa Cruz Biotechnology; SC-32233). The membranes were then incubated with corresponding secondary HRP-conjugated antibodies (Santa Cruz Biotechnology, USA) and the immune complexes were visualized by Pierce ECL (Thermo Scientific, USA) according to manufacturer’s protocol. Western blot experiments were performed as independent replicates.
9
HOXB9 Protein Expression Analysis
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The cells were lysed on ice and submitted to the western blot; the details are described in a previous study (17 (link)). The primary antibodies used in this study were listed as follows: HOXB9 (1:1000; Thermo Fisher Scientific, MA, US); β-tubulin (1:1000 dilution; Wanleibio Co., Ltd., Shenyang, China); HRP-conjugated antibodies (1:10,000; Santa Cruz Biotechnology, Inc., TX, USA).
10
Western Blot Analysis of Cellular Proteins
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Proteins were run on 8% SDS–polyacrylamide gels and blotted onto nitrocellulose membranes. Blots were blocked in 5% milk in PBST (1 × PBS and 0.1% Tween 20) and then incubated with the primary antibodies at 4 °C. The primary antibodies used were: GFP (1:1000, Novus), FAK (1:1000 Proteintech), paxillin (1:5000 BD Biosciences) and GAPDH (1:3000, SantaCruz). Membranes were cut prior antibody incubation. Visualization was performed using HRP-conjugated antibodies (Santa Cruz) after 1 h of incubation at room temperature, and a signal was detected using the ChemiDoc imaging system (Bio-Rad). Original blots are shown in Supplementary Information file.
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