The HSC-3 and HOEC cells were lysed in RIPA buffer, and total protein was quantified using bovine serum albumin (BSA, Boster, Wuhan, China). After being added with 5× loading buffer and boiled for 5 min, the proteins were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, U.S.A.). The membranes were blocked with 1× Blotto and incubated with primary antibodies (anti-KRT84 antibody, 1:1000, Abcam, U.S.A.; anti-GAPDH antibody, 1:1000, Abcam, U.S.A.) at 4°C overnight. After incubation with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (abcam, U.S.A.) at room temperature for 1.5 h, the membranes were developed with Western Lightning™ Chemiluminescence Reagent (PerkinElmer, U.S.A.). The intensity of protein was measured by using LabWorks™ (UVP, U.S.A.), with GAPDH as the internal control.
Horseradish peroxidase hrp labeled goat anti rabbit igg
Manufactured by Abcam
Sourced in United States
Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG is a secondary antibody conjugate that binds to rabbit immunoglobulin G (IgG) antibodies. The HRP enzyme label can be used to detect and quantify the presence of rabbit IgG in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Boster Bio (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti gapdh antibody, by Abcam (1 mentions)
Western lightning chemiluminescence reagent, by PerkinElmer (1 mentions)
0.45 μm pvdf membrane, by Beyotime (1 mentions)
Cfx connect, by Bio-Rad (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Lc3 rabbit polyclonal antibody, by Abcam (1 mentions)
Trizol rna extraction kit, by Thermo Fisher Scientific (1 mentions)
Mtt powder, by Merck Group (1 mentions)
Protein lysis solution, by Roche (1 mentions)
Ecl solution, by Thermo Fisher Scientific (1 mentions)
6 protocols using horseradish peroxidase hrp labeled goat anti rabbit igg
1
Western Blot Analysis of KRT84 Expression
2
Quantifying Phospho-Akt in BMSC Lysates
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Cell lysate (cat. no. P0013J, Beyotime, Shanghai, China) lysed 8.5 × 107 BMSCs in each group and centrifuged at 4 °C and 12000 rpm for 10 min. Protein concentrations were measured by a BCA protein concentration detection kit (cat. no. P0011, Beyotime, China), 55 μg of BMSC lysate samples from each group were subjected to SDS–PAGE on a 0.45 μm PVDF membrane (cat. no. FFP36, Beyotime, China). After the skimmed milk powder was sealed for 60 min, rabbit anti-human phospho Akt antibody (P-Akt, cat. no. ab8933, 1:250, Abcam, Cambridge, USA) and rabbit anti-human Akt antibody (cat. no. ab18785, 1:200, Abcam, USA) and incubated at 4 °C for 18 h; horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (cat. no. ab6721, 1:500, Abcam, USA) was incubated for 240 min at room temperature. β-actin (cat. no. ab5964, 1:400, Abcam, USA) was the internal reference control. An ultrasensitive ECL chemiluminescence kit (cat. no. P0018AS, Beyotime, Shanghai, China) and a Tanon 1600 gel image analysis system (Tanon. ltd., Shanghai, China) were used to detect protein expression. Image-Pro Plus 6.0 (Mediacy Cybernetics, Inc. Bethesda, MD, USA) analyzes the expression of each protein band.
3
Wnt3a Signaling Pathway Evaluation
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The terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay kit (cat. no. ab66110), antibodies against Wnt family member 3a (Wnt3a; cat. no. ab219412, 1/1,000), basic fibroblast growth factor (bFGF; cat. no. ab92337, 1/1,500), glycogen synthase kinase-3β (GSK-3β; cat. no. ab32391, 1/1,000), phosphorylated (p-)GSK-3β (cat. no. ab75745, 1/1,000), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; cat. no. ab8245, 1/1,000) and horseradish peroxidase (HRP)-labeled goat anti rabbit IgG (cat no. ab205719, 1/2,000) were obtained from Abcam. Rabbit antibody against vascular endothelial growth factor (VEGF; cat. no. sc-7269, 1/1,000) was obtained from Santa Cruz Biotechnology, Inc.; the polyvinylidene fluoride (PVDF) membrane (cat. no. IPVH00010) was purchased from MilliporeSigma; the Novex™ ECL Chemiluminescent Substrate Reagent kit (cat. no. WP20005), TRIzol® reagent (cat. no. 15596026), the RevertAid First Strand cDNA Synthesis kit (cat. no. K1621), TaqMan Pre-Amp Master Mix (cat. no. 4391128) and Lipofectamine™ RNAiMAX Transfection Reagent (cat. no. 13778030) were all purchased from Themo Fisher Scientific, Inc.; the ultrasensitive chemiluminescence imaging system (Chemi Doc™ XRS+) and fluorescence quantitative PCR (CFX Connect™) instruments were obtained from Bio-Rad Laboratories, Inc.
4
Licorice and Mesalazine Protect against Colitis
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DSS was purchased from Shanghai Yisheng Biotechnology Co., Ltd. Licorice was purchased from Anhui Guanghe Traditional Chinese Medicine Co., Ltd. Mesalazine sustained release granules were obtained from Aidifa Pharmaceutical Co., Ltd. The superoxide dismutase (SOD) kit, malondialdehyde (MDA) kit, and glutathione peroxidase (GSH‐PX) kit were from Jiancheng Biology Co., Ltd. The mitochondrial membrane potential kit, phosphate buffered saline (PBS), and ROS kit was purchased from Suolaibao Technology Co., Ltd. Enzyme‐linked immunosorbent assay (ELISA) kits were purchased from Jianglai Biotechnology Co., Ltd. glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) rabbit polyclonal antibody, Nrf2 rabbit polyclonal antibody, P62 rabbit polyclonal antibody, LC3 rabbit polyclonal antibody and horseradish peroxidase (HRP)‐labeled goat anti‐rabbit IgG were obtained from Abcam, PINK1 rabbit polyclonal antibody, Parkin rabbit polyclonal antibody, HO‐1 rabbit polyclonal antibody were purchased from Zhengneng Biotechnology Co., Ltd. roswell park memorial institute (RPMI)1640 (SH30027.FS) was from Hyclone Company, tissue dissociation kit (130‐110‐201).
5
Mesangial Cell Molecular Mechanisms
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The following cells, reagents, and antibodies were used in this study: human glomerular mesangial cells (ScienCell Research Laboratories); 4201 standard mesangial cell medium (MCM; ScienCell Research Laboratories); UA (Sigma, US); methyl thiazolyl tetrazolium (MTT) powder (Sigma, US); dichloro-dihydro-fluorescein diacetate (DCFH-DA) Reactive Oxygen Species (ROS) Detection Kit (Wuhan Beyotime); TRIzol RNA Extraction Kit (Invitrogen, US); GoTaq Quantitative Polymerase Chain Reaction (qPCR) Master Mix (Promega, US); rabbit anti-Akt, anti-p-Akt (p-Ser473), anti-mTOR and anti-p-mTOR (Ser2448) polyclonal antibodies (Cell Signaling Technology, US); rabbit anti-TGFβ1, anti-FN, anti-Bax, anti-Samd2/3, anti-Samd7 and anti-GAPDH polyclonal antibodies (Proteintech, US); and horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (Abcam, US).
6
Western Blot Analysis of Apoptosis and Cell Cycle Markers
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After cell treatment, the medium was discarded. The protein lysis solution (Roche) was added, and the total protein was separated. Then, 50 μg of the total protein was taken and sampled on a 12% polyacrylamide gel and electrophoresed (100 V, 2 h). Afterward, the protein was transferred to polyvinylidene fluoride (PVDF) membranes. After being blocked with 5% skimmed milk at RT for 1 h, the membranes were rinsed with TBST 3 times (10 min each time) and incubated with the primary antibodies (1:1000) including anti-Bad (ab32445), anti-bcl2 (ab32124), anti-Bax (ab32503), anti-Caspase3 (ab32351), anti-p21 (ab109520), anti-Cyclin D (ab16663), anti-CDK4 (ab108357), anti-Cyclin E (ab33911), anti-CDK2 (ab32147), anti-GRIM-19 (ab110240), anti-E-cadherin (ab16505), anti-Vimentin (ab92547), anti-N-cadherin (ab18203), anti-p-STAT3 (ab76315), anti-STAT3 (ab68153), and anti-HIF-1α (ab179483) overnight at 4℃. After washing the membranes with TBST, we incubated them with horseradish peroxidase (HRP)-labeled goat-anti-rabbit IgG at RT (ab205718, 1:2500) for 1 h. The above antibodies were all from Abcam (Cambridge, UK). The membranes were rewashed 3 times with TBST (10 min each time). Finally, the ECL solution (Invitrogen) was utilized for protein band development and imaging, and Image J was employed to analyze the gray value of each protein.
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