Equal amounts of cell lysates were separated by 12 % SDS-PAGE, and electrophoretically transferred to PVDF membrane. The membrane was blocked and probed with primary antibody (anti-ASK1, anti-phospho-ASK1, anti-p38, anti-phospho-p38, anti-ERK1/2, anti-phospho-ERK1/2, anti-ERK5, anti-phospho- ERK5, anti-JNK, anti-phospho-JNK from Cell Signaling Technology; anti-MEKK2, anti-NIS, anti-TSHR from Santa Cruz; anti-β-actin from Sigma) followed by HRP (horseradish peroxidase)-labeled goat anti-mouse IgG (Abcam) or HRP-labeled goat anti-rabbit IgG (Abcam). Chemiluminescence was used to analyze protein levels and β-actin was used as a protein loading control. Semi-quantitative analysis was conducted by using ImageJ 1.49v (National Institutes of Health, USA).
Hrp labeled goat anti rabbit igg
Manufactured by Abcam
Sourced in United Kingdom
HRP-labeled goat anti-rabbit IgG is a secondary antibody used in various immunoassay techniques. It is produced by immunizing goats with rabbit IgG and conjugating the resulting antibodies with horseradish peroxidase (HRP). This antibody can be used to detect and quantify the presence of rabbit primary antibodies in samples.
Automatically generated - may contain errors
Lab products found in correlation
Hrp labeled goat anti mouse igg, by Abcam (2 mentions)
Bca kit, by Beyotime (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti ask1, by Cell Signaling Technology (1 mentions)
Anti erk5, by Cell Signaling Technology (1 mentions)
Anti phospho erk5, by Cell Signaling Technology (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Anti p38, by Cell Signaling Technology (1 mentions)
Anti phospho jnk, by Cell Signaling Technology (1 mentions)
Anti jnk, by Cell Signaling Technology (1 mentions)
Anti phospho p38, by Cell Signaling Technology (1 mentions)
Anti phospho ask1, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Hematoxylin, by Carl Roth (1 mentions)
Impact dab peroxidase substrate kit, by Vector Laboratories (1 mentions)
Mirax midi scanner, by Zeiss (1 mentions)
Roti histol, by Carl Roth (1 mentions)
14 protocols using hrp labeled goat anti rabbit igg
1
Western Blot Analysis of Signaling Proteins
2
Immunohistochemical Insulin Detection in Pancreas
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Pancreatic sections (2 µm) were de-paraffinized and rehydrated in Roti-Histol (Carl Roth, Karlsruhe, Germany) and a decreasing serial solution of ethanol. Slides were heated in citrate buffer (10 mM citrate acid, 0.05% Tween 20) in a pressure cooker using a microwave for 30 min at 900 W followed by a cooling step (30 min, RT). Sections were incubated with blocking solution (5% BSA/TBST, 1 h, RT), followed by primary antibodies (1 h, RT). Rabbit anti-insulin (Abcam, Cambridge, UK, ab181547) was used as primary antibody. Hydrogen peroxide blocking was performed with a 0.3% solution (Carl Roth) for 15 min at RT, followed by incubation with HRP-labeled Goat Anti-Rabbit IgG (1 h, RT) (Abcam, ab205718). Before mounting with VectaMount Mounting Medium (H-5000), tissue sections were incubated with 3,3’-diaminobenzidine in chromogen solution (ImPACT DAB Peroxidase Substrate Kit, Vector Laboratories, Burlingame, CA, USA) and counterstained with hematoxylin (Carl Roth). Stained insulin in whole pancreatic sections were imaged with a MIRAX-MIDI Scanner (Carl Zeiss AG, Oberkochen, Germany), equally edited with Pannoramic Viewer and analyzed with ImageJ Software.
3
Immunohistochemistry Protocol for Tumor Tissue Analysis
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For IHC assays, tumor tissues were fixed with 4% formalin for 24 h at room temperature, paraffin-embedded and cut into 4-µm-thick sections. Subsequently, the slides were deparaffinized in xylene and rehydrated through a descending ethanol series. Antigen retrieval was performed with citrate buffer in a microwave oven. Subsequently, slides were blocked with 3% hydrogen peroxidase in methanol and 10% goat serum (Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature. Slides were incubated with CD4 (cat. no. ab183685; 1:1,000; Abcam), CD8 (cat. no. ab217344; 1:1,000; Abcam), TMED5 (cat. no. SRP08852; 1:1,1000; Tianjin Saier Biotechnology Co., Ltd.) and β-catenin antibodies (cat. no. ab223075; 1:1,000; Abcam) overnight at 4°C and then with HRP-labeled goat anti-rabbit IgG (cat. no. ab6721; 1:2,000; Abcam) for 30 min at room temperature. The slides were stained with DAB (1:50) as the chromogen and counterstained with hematoxylin for 1 min at room temperature. Slides were evaluated using an optic Olympus BX51 microscope at light field (Olympus Corporation; magnification, ×200) with a Micro-Publisher 3.3 RTV camera (Q Imaging Scientific & Industrial Camera; Teledyne Photometrics).
4
Curcumin Regulates Gastric Cancer Cell Signaling
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The human gastric cancer cell line, SGC-7901 was obtained from the Laboratory of Pathology, School of Basic Medical, Lanzhou University (Lanzhou, China),31 (link) and the cells were authenticated by STR. Cells were cultured in RPIM-1640 (HyClone, UT, USA) supplemented with 10% fetal bovine serum (FBS; Kibbutz Beit Haemek, Israel) and 1% penicillin/streptomycin (Sigma-Aldrich, MO, USA) in a humidified atmosphere of 5% CO2 at 37°C. Curcumin and a CCK-8 kit were purchased from Beijing Solarbio Science & Technology (Beijing, China).
Primary antibodies included: Anti-Shh (Abcam, Cambridge, UK), anti-Gli1 antibody (Abcam), anti-Foxm1 antibody (Abcam), anti-β-catenin antibody (Cell Signaling Technology, MA, USA), anti-E-Cadherin antibody (Cell Signaling Technology), anti-vimentin antibody (Cell Signaling Technology), anti-F-actin antibody (Abcam) and anti-β-actin antibody (Thermo Fisher Scientific, MA, USA). Secondary antibodies included: HRP-labeled goat anti-rabbit IgG (Abcam) and HRP-labeled goat anti-mouse IgG (Abcam). All the primary antibodies were diluted to 1:1000. The secondary antibodies were diluted to 1:5000.
Primary antibodies included: Anti-Shh (Abcam, Cambridge, UK), anti-Gli1 antibody (Abcam), anti-Foxm1 antibody (Abcam), anti-β-catenin antibody (Cell Signaling Technology, MA, USA), anti-E-Cadherin antibody (Cell Signaling Technology), anti-vimentin antibody (Cell Signaling Technology), anti-F-actin antibody (Abcam) and anti-β-actin antibody (Thermo Fisher Scientific, MA, USA). Secondary antibodies included: HRP-labeled goat anti-rabbit IgG (Abcam) and HRP-labeled goat anti-mouse IgG (Abcam). All the primary antibodies were diluted to 1:1000. The secondary antibodies were diluted to 1:5000.
5
Protein Expression Analysis of Osteoarthritis Chondrocytes
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Osteoarthritis chondrocytes were treated with RIPA lysis buffer (Beyotime), and the concentration of proteins was tested using the Advanced BCA Protein Assay Kit (Beyotime). After SDS-PAGE separation, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane for 1 h incubation with 1% bovine serum albumin (BSA, Sigma-Aldrich) and then washed with PBST. Primary antibodies were added and incubated overnight at 4°C to bind the target proteins, then secondary antibody was added and incubated for 1 h at 37°C. Primary antibodies were rabbit anti-WIF1, anti-βcatenin, anti-lamin B1, anti-βactin and anti-GAPDH (1 : 2000, 1 : 1000, 1 : 1000, 1 : 2000, 1 : 2000 respectively, Abcam, Cambridge, MA, USA). Secondary antibody was HRP-labeled goat anti-rabbit IgG (1 : 2000, Abcam). Blots were filmed by Vilber Lourmat (Marnela Vallée, France) and analyzed by Quantity One software (Bio-Rad, Hercules, CA, USA).
6
Western Blot Analysis of IRS and PI3K Signaling
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The expression of proteins was determined by western blot as previously described (18 (link)). The concentration of protein was detected using a BCA kit (Beyotime, Shanghai, China). Then, 30 μg of total protein was loaded onto 8% SDS polyacrylamide gels and transferred to the polyvinylidene fluoride membrane. The membrane then was blocked by 5% BSA (Sigma) for 1 h and incubated with primary antibodies at 4°C overnight. The next day, the membrane was washed with PBS for three times and then followed by incubation with HRP-labeled goat anti-rabbit IgG (Abcam, America) secondary antibody at 37°C for 1 h. Signals were detected by using the ECL detection kit (Merck Millipore) and normalized to the expression of GAPDH. The antibodies included Total insulin receptor substrate (IRS)-1 (ab52167, 1:1,000, Abcam), phosphorylated IRS-1 (p-IRS-1) at PY896 (sc-560, 1:500, Santa Cruz), total IRS-2 (ab52606, 1:1,000, Abcam), phosphorylated IRS-2 (p-IRS-2) at S731 (sc-1555-R,1:500, Santa Cruz), total PI3K (ab154598,1:1,000, Abcam), phosphorylated PI3K (p-PI3K) at P85 (ab191606, 1:1,000, Abcam), phosphorylated Akt (p-Akt) at Ser-473 (ab192623, 1:1,000, Abcam), GAPDH (ab9485, 1:1,000, Abcam). The absorbance of each band was quantitated using the Image Pro Plus Software.
7
CML Cell Autophagy and Apoptosis Regulation
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BEZ235 was purchased from Selleck, dissolved in dimethyl sulfoxide (DMSO) and stored at −20 °C. Chloroquine (CQ) was purchased from Sigma, dissolved in sterile double distilled water, and stored at −20 °C. 3-methyladenine (3-MA) was purchased from Selleck, dissolved in PBS and stored at −20 °C. Z-VAD-FMK was purchased from Beyotime and stored at −20 °C. The human CML cell line K562 was obtained from the Fujian Institute of Hematology, and the KBM7R cell line was purchased from the Harbin Institute of Hematology. Fetal bovine serum and RPMI 1640 medium were purchased from Gibco. Annexin V-FITC/PI kit was purchased from Becton Dickinson. MTS kit was purchased from Promega. Primary antibody AKT, P-AKT, S6K, P-S6K, Cleaved casepase-3, LC3I/II and HRP-labeled goat anti-rabbit IgG were purchased from Abcam. The RFP-GFP-LC3 double-labeled adenovirus was purchased from Hanbio. Cell constant temperature incubator (Thermo, USA), Refrigerated centrifuge (Eppendorf, Germany), Infinite M200 microplate reader (Tecan, Switzerland), FACS Calibur TM flow cytometry (Becton Dickinson, USA). Western blot electrophoresis, Semi-dry meter and Gel imager (Bio-Rad, USA). Hu-12A Transmission electron microscope (Hitachi, Japan), Laser scanning confocal microscope (Zeiss, Germany).
8
Protein Detection and Quantification using BCA
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The total amount of protein was detected by the bicinchoninic acid method (Beyotime, Shanghai, China). Proteins (20 μg) were separated by SDS-PAGE on 10% polyacrylamide gels and transferred electrophoretically to polyvinylidene fluoride membranes with the TGX Stain-Free FastCast Acrylamide Kit (Bio-Rad, California, USA). The membranes were blocked with 5% nonfat dry milk in TBS + 0.1% Tween-20 for 1 h and incubated overnight at 4 °C with the primary antibodies in bond primary antibody diluent (see Supplementary Table 2, Supplemental digital content 1, http://links.lww.com/WNR/A654 , for antibody information), followed by secondary antibodies [horseradish peroxidase (HRP)–labeled goat antimouse immunoglobulin (Ig)G, 1:5000, and HRP-labeled goat antirabbit IgG, 1:10 000, Abcam, Cambridge, UK] at room temperature for 1 h. The proteins were detected by the Bio-Rad ChemiDoc Touch Image System (Bio-Rad).
9
Western Blot Analysis of Exosomal Proteins
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After cell proteins were extracted, they were heated at 100 ℃ for 3 min to denature them. The protein was transferred to the negative control (NC) membrane, and the NC membrane was sealed in 5% skim milk powder [phosphate-buffered saline (PBS) preparation] at 37 ℃ for 2 h or 4 ℃ overnight. The primary antibody was then added to bind the target protein. The primary antibodies—CD9 (Abcam, Cambridge, UK), CD81 (Abcam, Cambridge, UK), Tumor susceptibility gene 101 (TSG101) (Abcam, Cambridge, UK), Heat Shock Protein 70 (HSP70) (Abcam, Cambridge, UK), ALG-2-interacting protein X (ALIX) (Abcam, Cambridge, UK), and flotillin-1 (Abcam, Cambridge, UK)—were incubated in a shaker at room temperature for 2 h or overnight at 4 ℃. The secondary antibody of horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (Abcam, Cambridge, UK) or goat anti-mouse (Abcam, Cambridge, UK) was incubated and incubated in a shaker for 1 h or overnight at 4 ℃. Diaminobenzidine (DAB) kit was used to develop color. ImageJ software (Bethesda, Maryland, USA) was used for gray value analysis of protein bands. Experimental results were recorded, and the NC film was dried, scanned, and preserved.
10
Nintedanib and Wnt3a Signaling Pathway
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Nintedanib (>99%) was purchased from HWRK Chem Co., Ltd. (Beijing, China). For each experiment, Nintedanib was freshly prepared by dissolving it in DMSO (Sigma-Aldrich, USA). Bleomycin (BLM) was acquired from Nippon Kayaku (Tokyo, Japan). Wnt3a was purchased from Peprotech (Texas, USA). TRIzol reagent and DEPC-treated water were obtained from Thermo Fisher Scientific corporation (Waltham, USA). RNase, DNase, and DNA Away H2O were purchased from Beyotime Biotechnology (Beijing, China). FastKing gDNA Dispelling RT SuperMix was obtained from Tiangen Biotech Co., Ltd. (Beijing, China). The inhibitor KX2-391, RIPA lysis buffer (middle) and the BCA kit were purchased from Beyotime Biotechnology (Beijing, China). The primary antibodies described in the study included anti-fibronectin, anti-collagen I, and anti-β-tubulin antibodies (Affinity Biosciences, USA); anti-GAPDH, anti-p(Y416)-Src, and anti-Src antibodies (Cell Signalling Technology, USA); anti-α-actin antibody (Santa Cruz Biotechnology, USA); anti-β-catenin and anti-lamin B1 antibodies (ProteinTech Group, China); and anti-p(Y654)-β-catenin antibody (Immunoway Biotechnology, China). The secondary antibodies HRP-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse IgG were from Abcam (Cambridge, UK).
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