SFAC (containing saponin constituents higher than 74.4%), escin Ia (C55H86O24, MW: 1131.26, purity ≥ 98%), escin Ib (C55H86O24, MW: 1131.26, purity ≥ 98%), escin IIa (C54H84O23, MW: 1101.23, purity ≥ 98%), escin IIb (C54H84O23, MW: 1101.23, purity ≥ 98%), escin IIIa (C55H86O23, MW: 1115.26, purity ≥ 98%) and escin IIIb (C55H86O23, MW: 1115.26, purity ≥ 98%) were obtained from Yinxing Pharmaceutical Technology Co., Ltd. (Nanjing, China) (Figure 1 ); β-aminopropionitrile (BAPN) was obtained from Sa'en Co., Ltd. (Shanghai, China); Transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α) were obtained from R & D Systems (Minneapolis, USA); TRIzol and Lipofectamine 2000 reagents were obtained from Invitrogen (Carlsbad, USA); E-cadherin and LOXL2 antibodies were purchased from Genetex (Irvine, USA); HIF-1α and vimentin antibodies were purchased from BioWorld (Georgia, USA); α-SMA antibody was purchased from Abcam (Cambridge, UK); GAPDH monoclonal antibody was purchased from KangChen Bio-tech (Shanghai, China); HRP-conjugated secondary antibodies were purchased from Abbkine (Redlands, USA); Fetal bovine serum (FBS) was obtained from PAA (Linz, Germany). Other analytical reagent grade chemicals were obtained from Sinopharm Chemical Reagent Co. Ltd (Nanjing, China).
Hrp conjugated secondary antibody
Manufactured by Abbkine
Sourced in United States, China
HRP-conjugated secondary antibodies are antibodies that have been labeled with the enzyme Horseradish Peroxidase (HRP). These secondary antibodies are used in various immunoassay techniques, such as ELISA and Western blotting, to detect and quantify the presence of target proteins or other biomolecules. The HRP enzyme allows for the generation of a colorimetric or chemiluminescent signal, which can be measured to determine the relative abundance of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Tumor necrosis factor α tnf α, by R&D Systems (1 mentions)
Vimentin, by Bioworld Technology (1 mentions)
Transforming growth factor β tgf β, by R&D Systems (1 mentions)
Hif 1α, by Bioworld Technology (1 mentions)
Gapdh monoclonal antibody, by Kangchen Biotech (1 mentions)
α sma antibody, by Abcam (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
E cadherin, by GeneTex (1 mentions)
P nf κbp65, by Immunoway (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Proteintech (1 mentions)
Traf6, by Immunoway (1 mentions)
Proliferating cell nuclear antigen (pcna), by Immunoway (1 mentions)
Proteinase inhibitor, by Beyotime (1 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Rabbit anti lc3, by Merck Group (1 mentions)
Rabbit anti p62, by Cell Signaling Technology (1 mentions)
Mouse anti β actin, by Abbkine (1 mentions)
Rabbit anti parp 1, by Santa Cruz Biotechnology (1 mentions)
6 protocols using hrp conjugated secondary antibody
1
Purification and Characterization of Escin Derivatives
2
Western Blot Analysis of Cellular Proteins
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The cells were washed with PBS before being lysed in lysis buffer that can extract both cytoplasm proteins and nucleoproteins. The sample was stored at −80°C. Samples containing an equal amount of proteins were mixed with 5x SDS loading buffer and electrophoresed on a 10% SDS-PAGE gel. The proteins were then transferred onto PVDF membrane (Millipore). The membranes were blocked and probed with antibodies anti-NFAT2 (CST), TRAF6 (Immunoway), p-NF-κBp65 (Immunoway), PCNA (Immunoway), p65 (Immunoway), and β-actin (Proteintech group). The membranes were incubated with specific primary antibodies overnight at 4°C at a 1 : 1000 dilution. Subsequently, the membranes were washed with TBS/T for 15 min, three times, and incubated for 1 h with HRP-conjugated secondary antibodies (Abbkine). The protein was visualized with chemiluminescence, and a densitometric scanner was used to determine the density of the band. All experiments were repeated at least three times independently.
3
Protein Extraction and Subcellular Fractionation
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Total proteins were extracted with RIPA lysis buffer (Beyotime) containing 1% proteinase inhibitors (Beyotime). The mitochondria isolation followed the instructions of the Cell Mitochondria Isolation Kit (Beyotime). Nuclear and cytoplasmic proteins were isolated using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime). The protein concentrations were quantified using a BCA Protein Assay Kit (Beyotime). Equal amounts of protein samples were subjected to 10–12% SDS-PAGE separation and then transferred to the polyvinylidene difluoride membrane (Millipore, Boston, MA, USA). Subsequent procedures were performed as described previously [38] (link). The following antibodies were used: rabbit anti-PARP-1 (1: 500, Santa Cruz, CA, USA), rabbit anti-AIF (1:1000, Abcam), rabbit anti-LC-3 (1:1000, Sigma-Aldrich), rabbit anti-p62 (1:1000, Cell Signaling Technology, MA, USA), mouse anti-PAR (1:1000, Enzo Life Sciences), mouse anti-β-actin (1:5000, Abbkine), rabbit anti-COX Ⅳ (1:1000, Proteintech, Wuhan, China), mouse anti-Histone H3 (1:3000, Abbkine), HRP-conjugated secondary antibodies (1:5000, Abbkine). Protein bands were detected by enhanced chemiluminescence kit (Thermo, USA).
4
Protein Isolation and Quantification from Visceral Adipose Tissue
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The total proteins from 100 mg visceral adipose tissue samples were isolated by radioimmunoprecipitation buffer (G-Biosciences, St. Louis, MO, USA) followed by their quantification using a bicinchoninic acid protein assay kit (G-Biosciences, USA). In brief, 30 µg denatured protein was resolved on 10% Tris–glycine gel and transferred to a polyvinylidene fluoride membrane (Bio-Rad, USA). The non-specific binding sites on the membrane were blocked with 5% skimmed milk. The membrane was incubated with corresponding primary antibodies (Abbkine, USA) at 1:700 dilutions overnight at 4°C, and after washing the membrane was treated with an HRP-conjugated secondary antibody (Abbkine, USA) for 2 h. The detection of signals was recorded by the use of an ECL Reagent Kit (Thermo Fisher Scientific, USA) in the gel imaging system (Thermo Fisher Scientific ECL Imager). The protein band density was measured and compared using the ImageJ software.
5
Recombinant NC8 aCD11c Protein Expression
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The recombinant NC8 (pSIP409-aCD11c) strains were cultured using MRS medium to examine the expression of aCD11c, a NC8 (pSIP409) strain was also included as negative control. To induce aCD11c expression, SppIP, was added into the medium at a final concentration of 50 ng/ml when the cultures reached a 600 nm absorbance (A600) of 0.3. After induction at 30°C for 10 h, the cultures were adjusted to the same A600 values, harvested by centrifugation at 5,000 ×g for 10 min at 4°C, and then washed twice times using 500 μl of cold, phosphate-buffered saline (PBS) and resuspended in 1 ml of PBS containing 1 mM CaCl 2 , 1.1 M sucrose, 0.5 M MgCl 2 , 100 μg of RNase (Sigma Chemicals, USA) , 50 μg of DNase (Sigma), and a cocktail of anti-proteases (Complete; Roche Molecular), and then 5 mg of lysozyme (Sigma) plus 100 μg of mutanolysin (Sigma) were also added. The digestion reactions were performed for 30 min at 37°C with shaking at 120 rpm and then centrifugation for 30 min at 10,000 ×g. The supernatants were acquired [20] and the protein samples from cell wall fractions were then subjected to western blotting assay using His-tag antibody (CWBIO, China) as primary antibody and HRPconjugated secondary antibody (Abbkine, USA). Visualization of the immunobinding was conducted by enhanced chemiluminescence (ECL) using an ECL Plus detection kit (Thermo Scientific).
6
Quantification of m6A RNA Modifications
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In short, mRNA was divided into different concentration gradients then spotted to Hybond-N + membrane, followed by UV crosslinking at UV 254 nm with 0.12 J/cm2. After blocking in TBST buffer containing 5% non-fat milk for 1 h, the membrane was incubated with 1:2000 diluted anti-m6A antibody (Cat. No. 202 003, Synaptic Systems, Göttingen, Germany) 2 h at 4 °C. The membrane was washed with TBST three times and then incubated with HRP conjugated secondary antibody (1:5000, Abbkine Scientific Co., Ltd., California, USA, Cat. No. A21010) for 1 h. And visualized by using ImmobilonTM Western HPR Substrate Luminol Regeant (Merck Millipore).
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