ELISA was used to identify positive clones of hybridomas and measure the titers of pAb and mAb generated. ELISA plates were coated with 0.4 μg recombinant proteins/well. The plates were incubated at 37°C in a wet box for 2 hours. After washing, 1% BSA-PBS was added to the wells to block nonspecific binding of the ELISA plates for 2 h. For serum titer determination, immune and preimmune sera were serial-diluted prior to the addition of ELISA wells. Horseradish peroxidase-conjugated goat-anti-rabbit IgG or goat-anti-mouse IgG (Beyotime) was used as the secondary antibody in ELISA. The optical density (OD) of the reaction was measured at 450 nm in a microplate reader (BioTek, America). For the identification of the positive hybridoma clones, 100 μL culture was used as the source of the primary antibodies in ELISA. Positive (post-immune serum at 1:1,000 dilution) and negative (pre-immune serum or media from SP2/0 cell cultures) controls were included in each ELISA test. The hybridoma clones were identified as positive when the OD values obtained were at least three times greater than those from negative cultures.
Horseradish peroxidase conjugated goat anti rabbit igg
Manufactured by Beyotime
Sourced in China
Horseradish peroxidase-conjugated goat anti-rabbit IgG is a secondary antibody used in various immunoassay techniques. It consists of goat-derived antibodies specific to rabbit immunoglobulin G (IgG), which are conjugated to the enzyme horseradish peroxidase. This conjugate can be used to detect and quantify the presence of rabbit IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg, by Beyotime (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (2 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Astragalin, by Chengdu Must Bio-Technology (1 mentions)
Hyperoside, by Chengdu Must Bio-Technology (1 mentions)
Srebp1, by Beyotime (1 mentions)
Streptozotocin (stz), by Merck Group (1 mentions)
Pepsin, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis solution, by Beyotime (1 mentions)
Phenylmethanesulfonyl fluoride, by Beyotime (1 mentions)
Bcl2 associated x (bax), by Beyotime (1 mentions)
Bcl 2, by Beyotime (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Pakt t308, by Cell Signaling Technology (1 mentions)
Bicinchoninic acid bca method, by Beyotime (1 mentions)
Nedd9, by Cell Signaling Technology (1 mentions)
21 protocols using horseradish peroxidase conjugated goat anti rabbit igg
1
ELISA-based Hybridoma and Antibody Titer Analysis
2
Investigating TCDD-Induced CYP1A1 and AhR Expression
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Cardiomyocytes were pre-treated with various concentrations of TCDD (0, 0.1, 1, 5 or 10 nM) for 24 h. The cells were then rinsed with ice-cold PBS, and the cytoplasmic and nuclear proteins were lysed using radioimmunoprecipitation buffer (HEART Biological Technology Co., Ltd., Xi'an, China). A total of 25 µg protein was separated from each treatment group using 12% SDS-PAGE, and the separated proteins were then electrophoretically transferred onto polyvinylidene difluoride membranes. The membranes were blocked in 0.75 g skim milk dissolved in 15 ml tris-buffered saline with Tween-20 (TBST) and probed with primary polyclonal rabbit anti-human antibodies against CYP1A1 (catalog no. sc-39397; 1:500), AhR (catalog no. sc-166586; 1:800) and β-actin (catalog no. sc-130656; 1:400; Santa Cruz Biotechnology, Inc.) at 4°C overnight. The primary antibodies were then discarded and the membranes were washed three times with TBST and incubated with the appropriate horseradish peroxidase-conjugated goat anti-rabbit IgG (catalog no. A0216; 1:400; Beyotime Institute of Biotechnology, Haimen, China) for 2 h with shaking at room temperature. The bands were visualized using enhanced chemiluminescence according to the manufacturer's instructions(EMD Millipore, Billerica, MA, USA).
3
Antidiabetic Potential of Mulberry Leaves
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Mulberry leaves were collected from Longling county (Baoshan, China) and identified to be the leaves of Mours alba by Prof. Baolian Guo, the Institute of Medicinal Plant Development, Peking Union Medical College. The insulin detection kit was purchased from Wuhan Miting Biotechnology Co., Ltd. (Wuhan, China). Assay kits for triglyceride (TG), total cholesterol (T-CHO), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Streptozotocin was purchased from Sigma Co., (Saint Louis, MO, USA). Standard mouse diet was purchased from Mouse One Mouse Two Biological Co., Ltd. The antibodies for AMPK, SREBP1, ACC, CPT1A, FAS, and horseradish peroxidase-conjugated goat-anti-rabbit IgG were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Other chemicals were supplied by Damao Co., Ltd. (Tianjin, China). Standards of rutin, hyperoside, and astragalin were purchased from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China).
4
In vitro Digestion of HBD3 Milk
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HBD3 milk was skimmed and used for in vitro analysis according the previously described protocol with slight modification [14 (link), 15 (link), 24 (link)]. Simulated gastric fluid was prepared by dissolving human pepsin (800–2500 U/mg, Sigma, St. Louis, MO, USA) into 0.03 mol/L NaCl solution and adjusted pH value to 2 with HCl, and the final concentration of pepsin is 500 mg/ml. Every 500 μl skim milk were mixed with 10 μl simulated gastric fluid, and incubated in a water bath at 37°C for 0, 1, 5, 15, 60, 120 min, respectively. The enzymatic reactions were stopped by adjustment of pH to 7 with 1 mol/L NaOH. The digestion products at 0, 1, 5, 15, 60, 120 min were separated by 10% Tricine-SDS-PAGE. Proteins were visualized by Coomassie Brilliant Blue staining. In western blot analysis, a rabbit polyclonal antibody against HBD3 (1:1000 dilution, Sigma, St. Louis, MO) was used as primary antibody and horseradish peroxidase-conjugated goat anti-rabbit IgG as the second antibody (1:1000 dilution, Beyotime, Jiangsu, China).
5
Quantitative Protein Analysis in Infected Cells
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Total protein isolation was carried out from infected cells by employing the phenylmethanesulfonyl fluoride (Beyotime) mixed with RIPA lysis solution (Beyotime). Protein concentration was measured by the BCA Protein Assay Kit (Beyotime). 25 μg protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Merk Millipore, Billerica, MA, USA). After blocked with 5% skimmed milk, membranes were incubated with the antibodies against RUNX1 (1:400; Affinity, Changzhou, China) or β-actin (1:1000; Santa Cruz) at 4 °C overnight and then with the secondary antibodies horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000; Beyotime) or goat anti-mouse IgG (1:5000; Beyotime). Protein blot bands were visualized with WD-9413B gel imaging system (Liuyi biotechnology, Beijing, China).
6
Baicalein Modulates Signaling Pathways
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BxPC-3 and PANC-1 cells treated with Baicalein, inhibitors, or siRNAs were lysed in the RIPA buffer (Beyotime Biotechnology). The concentrations of the total proteins were measured using the Bicinchoninic acid (BCA) method (Beyotime Biotechnology). Protein samples (20 μg) were subjected to SDS-PAGE and transferred to nitrocellulose membranes by electroblotting. The membranes were incubated with the primary antibodies and then the corresponding secondary antibodies (horseradish peroxidase-conjugated goat anti-Rabbit IgG or goat anti-mouse IgG at 1:1000 dilution, Beyotime Biotechnology). The signals were detected using Super ECL Detection Reagent (High-sig ECL Western Blotting Substrate, 180-5001, Tanon, Shanghai, China). Primary antibodies used: NEDD9 (#4044, 1:500), Akt (#4691, 1:1000), p-Akt S-473 (#4060, 1:1000), p-Akt T-308 (#2965, 1:1000), ERK1/2 (#9102, 1:1000), p-ERK1/2 (#9101, 1:1000), PDK1(#3062, 1:1000), P21(#8831, 1:1000), P27(#3686, 1:1000) and cleaved caspase-9 (#7237, 1:1000) from Cell Signaling Technology Inc. (Danvers, MA, USA), Bax (AB026, 1:1000) and Bcl-2 (AB112, 1:1000) from Beyotime Biotechnology (Shanghai, China).
7
Western Blot Analysis of Apoptosis Markers
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A549/DDP cells were collected from each drug-treated group. Protein extraction was performed using RIPA lysis buffer (Beyotime, China), and protein concentration was quantified with a bicinchoninic acid protein assay kit (Bio-Rad Laboratories, Hercules, USA). The protein samples were separated by SDS-PAGE and transferred onto PVDF membranes (The PVDF membrane presenting blots with full-length marker is over-sized for the WB transfer chamber. To adjust the PVDF membrane into the WB transfer chamber, we did cut it to the small-sized ones which can indicate 10–70 KDa. Also, it could blot proteins more stable). The membranes were then blocked with 5% non-fat milk for 2 h. Monoclonal rabbit antibodies against Caspase-3 (ab32351, Abcam, UK), Bax (ab32503, Abcam, UK), and GAPDH (ab181602, Abcam, UK) were added separately for incubation overnight at 4 ℃. Following washes with 0.1% TBST, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Beyotime, China) for 2 h. Protein bands were assessed with an ECL detection kit (Pierce, Rockford, USA).
8
Western Blot Analysis of SPINK7 and SP2
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Serum samples were separated using 12% SDS-PAGE followed by electrotransfer to PVDF membrane (GE Healthcare, USA). The membrane was blocked with 5% BSA in TBST buffer (50 mM Tris-HCl, 500 mM NaCl, and 0.2% Tween 20, pH 8.0) for 2 h, and then incubated with rabbit anti-SPINK7 or anti-storage protein 2 (SP2) for 1.5 h at 25°C. After washing three times with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Beyotime, China) at 25°C for 1h and detected using ECL reagent (GE Healthcare, USA).
9
Protein Expression Analysis in Spinal Tissue
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L4-5 spinal segmental tissue or cultured cells were homogenized for 30 min on ice in RIPA lysis buffer (Beyotime, China) containing a mixture of phosphatase and proteinase inhibitors. The sample was centrifuged at 4 °C at 16,000 rpm for 20 min, and then the protein supernatant was collected. The concentration of total protein was measured by BCA protein assay (Beyotime, China). Equal amounts of total proteins (30 µg/lane) were separated on a 10% SDS-PAGE gel and then transferred onto NC membranes. The membrane was blocked with 5% nonfat milk in PBS at RT for 1 h and incubated with antibodies against GFAP (1:1000, rabbit, Abcam), IBA1 (1:500, rabbit, Abcam), NF‐κB (1:500, rabbit, Abcam) and GAPDH (rabbit, 1:5000; CST) overnight at 4 °C. This membrane was washed and further incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000, Beyotime) at RT for 1 h. The signals were developed using Pierce ECL western blotting Substrate. The intensity of blots was quantified with densitometry using Alpha EaseFC software (Bio-Rad, USA). All protein bands were normalized to GAPDH.
10
Western Blot Analysis of Cellular Proteins
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Proteins were extracted from cells by lysing in RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors. After protein quantification, cell lysates were suspended in 5x loading buffer and then equal amounts of proteins were subjected to migration on 4%–20% polyacrylamide linear gradient gels, then transferred to a polyvinylidene fluoride membrane (Millipore). The membranes were blocked by nonfat milk (5%) for 60 min and then incubated with primary antibodies overnight at 4°C. Horseradish peroxidase-conjugated goat antirabbit IgG (1 : 5,000, Beyotime) was used as a secondary antibody for 1 hr at room temperature. Immunoreactive bands were detected using an enhanced chemiluminescent detection reagent (Millipore). The signal intensity was measured using a Bio-Rad XRS chemiluminescence detection system (Bio-Rad, Hercules, CA, United States).
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