Sodium alginate (300 – 400 mPa⋅s) was purchased from Chemiz (Malaysia). Gold (III) chloride trihydrate (HAuCl4·3H2O), 3,3′-anthracene-9,10-diyldipropanoic acid (ADPA), 1,3-diphenylisobenzofuran (DPBF), triton X-100 and mannitol were purchased from Sigma-Aldrich (Germany). Sodium borohydride (NaBH4), dimethyl sulfoxide (DMSO) and horseradish peroxidase (HRP) substrate were supplied by Merck (Germany). M-PERTM mammalian protein extraction reagent was purchased from Thermo Fischer Scientific (USA). Thiazolyl blue tetrazolium bromide (MTT) and ascorbic acid were obtained from BioBasic (Canada). Hexadecyltrimethylammonium bromide (CTAB) was purchased from Sangon Biotech (China). Silver nitrate (AgNO3) was acquired from Bendosen (Malaysia). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Nacalai Tesque (Japan). Fetal bovine serum (FBS) was purchased from GIBCO™ (USA). Apoptosis detection kit was supplied by Elabscience (USA). Unless stated otherwise, all reagents were prepared using Milli-Q ultrapure water.
Horseradish peroxidase hrp substrate
Manufactured by Merck Group
Sourced in United States
Horseradish peroxidase (HRP) substrate is a colorimetric reagent used to detect and quantify the presence of HRP in various biological assays. It serves as an indicator, reacting with HRP to produce a colored product that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Mannitol, by Merck Group (1 mentions)
M pertm mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Apoptosis detection kit, by Elabscience (1 mentions)
Thiazolyl blue tetrazolium bromide mtt, by Bio Basic (1 mentions)
Dulbecco s modified eagle s medium dmem, by Nacalai Tesque (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Peroxidase conjugated mouse anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Gel pro analyzer 4, by Media Cybernetics (1 mentions)
Radio immunoprecipitation assay ripa lysis buffer, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by GE Healthcare (1 mentions)
Anti flag rabbit monoclonal antibody, by Cell Signaling Technology (1 mentions)
Sds page loading buffer, by Beyotime (1 mentions)
Eblot l1, by GenScript (1 mentions)
Mouse monoclonal anti ha antibody, by Merck Group (1 mentions)
Semi dry electrotransfer, by Bio-Rad (1 mentions)
Anti phospho p44 p42 mapk anti ptepy, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
11 protocols using horseradish peroxidase hrp substrate
1
Alginate-Based Nanoparticle Synthesis and Characterization
2
Phosphorylated eIF2α Detection in DENV-2 Infection
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Poly(I:C)-treated or DENV-2-infected CCL-125 cells were collected and lysed using lysis buffer (1 mM of DTT and 1× protease inhibitor in HEPES-NaCl-Glycerol-Triton X 100 (HNTG) buffer). The cell lysates were run on a 10% polyacrylamide gel. The proteins were transferred to a membrane (Millipore Immobilon-P, Merck Millipore, Burlington, MA, USA), which was incubated with anti-eIF2α (phospho S51) primary antibody (Abcam, Cambridge, UK) for 12 h. The peroxidase-conjugated mouse anti-rabbit IgG (Jackson Laboratory, Bar Harbor, ME, USA) secondary antibody was reacted with horseradish peroxidase (HRP) substrate (Merck Millipore, Burlington, MA, USA) Actin antibody (Jackson Laboratory, Bar Harbor, ME, USA) was used as a loading control.
3
Protein Isolation and Western Blotting Protocol
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Proteins were isolated from the liver and gut tissue using radio-immunoprecipitation assay (RIPA) buffer purchased from Beyotime Biology (Shanghai, China) containing a protease and phosphatase inhibitor cocktail purchased from Beyotime Biology. After pulverization using a bead mill homogenizer purchased from Jingxin (Shanghai, China), the protein was quantified using a bicinchonininc acid (BCA) kit purchased from Beyotime Biology. Consequently, 40 mg of protein was separated on a polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane purchased from Millipore. After incubation with 5% bovine serum albumin (BSA) purchased from Service Biology (Wuhan, China) blocking for 1 h, membranes were incubated overnight, with the primary antibody in a 4 ℃ refrigerator. The secondary antibody was incubated for 1 h then, and the bands were detected using a chemiluminescent horseradish peroxidase (HRP) substrate purchased from Millipore. Densitometric band analysis was performed using a Gel-Pro Analyzer 4 from Media Cybernetics Corporation (Rockville, MD, USA).
4
Western Blot Analysis of Cell Signaling Proteins
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Total protein from tissues and cell lines was extracted by Radio Immuno Precipitation Assay (RIPA) lysis buffer (Beyotime, Shanghai, China), added to 10% sodium sulfate polyacrylamide gel (SDS-PAGE) and transferred onto 0.22 μm polyvinylidene fluoride (PVDF) membranes (Millipore, Massachusetts, USA). Subsequently, specific antibodies against p38, CDK1, CDK2, CDK4, vimentin, cyclin D1, and GAPDH were used [Supplementary Table 3, http://links.lww.com/CM9/B556 ]. The membranes were blocked with 5% skim milk powder and incubated with primary antibodies at 4°C overnight. After that, a chemiluminescence horseradish peroxidase (HRP) Substrate (Millipore, Massachusetts, USA) system was used to visualize the protein blots.
5
Western Blot Protein Quantification
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The collected cell lysates were centrifuged at 12,000× g for 30 min at 4 °C. The protein concentrations were estimated by the Bradford method (Bio-Rad, Hercules, CA, USA). Equivalent amounts of proteins from each group were separated with 8–12% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (GE, Amersham, UK). After protein transfer to the membrane, the membrane was blocked with 5% nonfat milk in Tris buffered saline (TBST) at room temperature for 1 h and incubated overnight with specific primary antibodies at 4 °C. After washing with TBST, the blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h, and then bands were visualized using enhanced chemiluminescent Horseradish Peroxidase (HRP) substrate (Millipore, Billerica, MA, USA). To ensure equal protein loading, GAPDH was used as an internal control. Densitometric analysis was carried out using ImageJ software (NIH, Bethesda, MD, USA).
6
Western Blotting of Protein Targets
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Western blot analysis was performed as described previously [20 (link)]. Protein samples were resolved by SDS-PAGE, followed by electroblotting to polyvinylidene difluoride (PVDF) membranes. The blots were probed with anti-HA mouse monoclonal antibody (1:1000, Cell Signaling), anti-Flag rabbit monoclonal antibody (1:1000, Cell Signaling), ZDHHC5-specific rabbit polyclonal antiserum (1:100, Sigma), and GOLGA7-specific rabbit polyclonal antiserum (1:1000, Abclonal). Peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (H + L) antibody was used as the secondary antibody. The signals were detected with a chemiluminescent horseradish peroxidase (HRP) substrate (Millipore).
7
Quantitative Yeast Cre Protein Analysis
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Yeast cell pellets (10 OD600 units) were resuspended and incubated with 200 µl NaOH (100 mM) at room temperature for 5 min. The NaOH-treated yeast cells were harvested via centrifugation (10,000 g for 2 min) and were subjected to cell lysis treatment via boiling for 10 min in 100 µl of 2x SDS-PAGE loading buffer (Beyotime, Cat number: P0015B). Equivalent levels of protein loading for whole cells were determined on the basis of the OD600 of the cell culture (0.1 units per lane) and confirmed by Coomassie's brilliant blue staining of parallel gels. Proteins were separated by reducing 12 % SDS-PAGE and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Millipore) via eBlotTM L1 (Genscript) transfer system. Cre enzymes were detected by the Cre-specific monoclonal antibody (Invitrogen, Cat number: MA5-27870) and alkaline phosphatase-linked anti-mouse IgG peroxidase antibody (Sigma-Aldrich, Cat number: A2304). Immunoreactive antigens were detected by chemiluminescence using horseradish peroxidase (HRP) substrate (Millipore, Cat number: WBKLS0100).
8
Western Blot Analysis of Protein Samples
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Western blot analysis was performed as described previously [30 (link)]. Protein samples were resolved by SDS-PAGE, followed by electroblotting to polyvinylidene difluoride (PVDF) membrane. The blots were probed with anti-HA mouse monoclonal antibody (1: 1000, Sigma, Sigma-Aldrich LLC., Santa Clara, CA, USA) or anti-Flag rabbit monoclonal antibody (1: 1000, Abbkine, Abbkine Scientific Co., Ltd. Wuhan, China). Peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (H+L) antibody was used as the secondary antibody. The signals were detected with a chemiluminescent horseradish peroxidase (HRP) substrate (Millipore, Millipore Corporation, Billerica, MA, USA).
9
Soybean Protein Extraction and Western Blotting
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Protein was extracted from flg22-treated soybean leaf tissues in an extraction buffer (50 mm Tris-MES, pH 8, 0.5 m Suc, 1 mm MgCl2, 10 mm EDTA, and 5 mm DTT as described [49 (link)]. For Western blotting, proteins were separated by SDS-PAGE (10% acrylamide gel) and transferred to PVDF membranes (Millipore, Burlington, MA, USA) by semidry electrotransfer (Bio-Rad) followed by incubation with the anti-phospho-p44/p42 MAPK (anti-pTEpY) diluted at 1:2000 (Cell Signaling Technology, Danvers, MA, USA). The bands were visualized using horseradish peroxidase (HRP) substrate (Millipore). Coomassie Blue–stained gel (CBS) was used as an equal loading control.
10
Immunoblot and Immunoprecipitation Protocols
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For immunoblot analysis, cells were lysed using cold radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology) supplemented with a complete protease inhibitor cocktail (Roche) and the phosphatase inhibitor PhosSTOP (Roche) according to the manufacturer's instructions. Protein concentrations of the extracts were measured using a bicinchoninic acid (BCA) assay (Beyotime Biotechnology) and equalized with lysis buffer. Equal amounts of the extracts were loaded and subjected to SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore), and blotted with the chemiluminescence horseradish peroxidase (HRP) substrate (Millipore). For IP, cells were lysed using lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100) and a complete protease inhibitor cocktail. Extracts were incubated overnight with FLAG M2 Affinity Gel, anti-HA-Agarose, or appropriate antibodies plus protein A/G beads. Beads were washed, eluted, and then operated as immunoblot analysis.
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