The total protein of uterine tissue was extracted by the lysate instructions (containing phenylmethanesulfonyl fluoride [PMSF], Beyotime, Shanghai, China) and detected using a BCA protein assay kit (Tiangen Biotech, China). The sample size was 50 μg protein per sample. Samples were separated by electrophoresis on polyacrylamide gels, and were subsequently transferred to nitrocellulose membranes. The membranes were incubated in 5% skimmed milk powder for 2 h, and then washed with Tris buffered saline Tween (TBST) (pH 7.6) (3×10 min), followed by addition of the primary antibody (polyclonal rabbit anti GHR, 1:300 (diluted by primary antibody dilution buffer, Beyotime, China), BIOSS, Beijing; monoclonal anti Actin, 1:1,000, Beyotime, China) and incubated at 4°C overnight. After washing with TBST, the nitrocellulose membranes were then incubated with anti-rabbit IgG antibody (1:3,000 [diluted by secondary antibody dilution buffer, Beyotime, China]; CWBIO) and anti-mouse IgG (1:3,000 [diluted by secondary antibody dilution buffer, Beyotime, China]; CWBIO) for 2.5 h at 37°C. Following washing with TBST, membranes were immersed in a high-sensitivity luminescence reagent (BeyoECL Plus, Beyotime, China), exposed to film using FusionCapt Advance FX7 (Fusion FX, OSTC), and analyzed using Ipp 6.0 (Image Pro-Plus 6.0, Media Cybernetics, USA).
Secondary antibody dilution buffer
Manufactured by Beyotime
Sourced in China
Secondary antibody dilution buffer is a solution used to dilute secondary antibodies in immunological experiments. It maintains the integrity and activity of the secondary antibody during the dilution process.
Automatically generated - may contain errors
Lab products found in correlation
Primary antibody dilution buffer, by Beyotime (12 mentions)
Beyoecl plus, by Beyotime (2 mentions)
Immunol staining blocking buffer, by Beyotime (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Western blocking buffer, by Beyotime (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (2 mentions)
Polyvinylidene difluoride pvdf membrane, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Apoptosis and necrosis assay kit, by Beyotime (1 mentions)
Sds page gel quick preparation kit, by Beyotime (1 mentions)
Cell lysis buffer for western and ip, by Beyotime (1 mentions)
Polyvinylidene fluoride pvdf membrane, by Beyotime (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Blocking buffer, by Beyotime (1 mentions)
Ix71 fluorescence microscope, by Olympus (1 mentions)
Goat anti mouse lgg hrp sc 2005, by Santa Cruz Biotechnology (1 mentions)
Trypsin, by Promega (1 mentions)
Bradford protein assay kit, by Beyotime (1 mentions)
Cell counting kit 8 cck 8 kit, by Beyotime (1 mentions)
Dapi solution, by Solarbio (1 mentions)
18 protocols using secondary antibody dilution buffer
1
Quantification of Uterine Protein Expression
2
Quercetin and Rutin Cytotoxicity Analysis
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Quercetin, rutin, dimethyl sulfoxide (DMSO) and 5-fluorouracil were purchased from Shanghai Kaiyang Biotechnology Company (Shanghai, China). Acetonitrile (HPLC), RPMI Medium 1640, and fetal bovine serum (FBS) were products of Gibco Life Technologies (NY, USA). The Apoptosis and Necrosis Assay Kit was purchased from the Beyotime; OLYMPUS IX73. Guava easyGyte; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), Cell lysis buffer for Western and IP, the SDS-PAGE Gel Quick Preparation Kit, Protein Marker, Polyvinylidene Fluoride (PVDF) Membrane, Blocking Buffer, Primary Antibody Dilution Buffer, Secondary Antibody Dilution Buffer, and BeyoECL Plus were purchased from the Beyotime Institute of Biotechnology (Shanghai, China).
3
Immunofluorescence Analysis of Nrf2 in Bovine Mammary Cells
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The bovine mammary epithelial cells (MAC-T) were seeded in 24-well plates and grown until 50% confluence. Cells were then fixed with 10% formalin and blocked with Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) followed by washing with TBST buffer (Triton X-100, 50 mM Tris, 0.15 mM NaCl, pH 7.6 containing 0.1% and Tween-20) three times. Cells were then incubated with a primary antibody for Nrf2 at a dilution of 1:200 (Abcam, Cambridge, MA, USA) at 4 °C for 12 h. After washing with TBST buffer, the cells were incubated with an FITC-labeled IgG diluted 1:1000 (Beyotime, Shanghai, China) in a special Secondary Antibody Dilution Buffer, (Beyotime, Shanghai, China). The nuclei of cells were stained with PI (Beyotime, Shanghai, China) for 15 min. The cells were viewed and analyzed using an Olympus fluorescence microscope (IX71, Tokyo, Japan).
4
Western Blot Analysis of McIL-17-3 Protein
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Protein samples were extracted from hemocytes with RIPA lysis buffer (Beyotime), and then the concentration was detected with a BCA kit. After isolation by SDS-PAGE, the protein was transferred to PVDF membranes with sealing by 5% skim milk powder in TBST (20 Mm Tris-HCl, 150 mM NaCl, 1% Tween-20, pH 8.0), followed by the antibody against McIL-17-3 incubation overnight. Subsequently, the membranes were incubated with a diluted solution of goat anti-mouse IgG antibody and alkaline phosphatase conjugate (Thermo Fisher Scientific, Cat. No.: 31324) in the secondary antibody dilution buffer (Beyotime, P0258) for 3 h. Finally, the immunoreactive proteins were detected by the ECL detection system.
5
Proteomic Analysis of Fetal Bovine Serum
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Fetal bovine serum (KGY009), incomplete culture medium (KGM1640SF), and the Nuclei and Cytoplasm Protein Extraction Kit (KGP150) were supplied by KeyGen Biotech (Nanjing, China). The Bradford protein assay kit, Cell Counting Kit-8 (CCK-8 Kit), western blocking buffer, primary antibody dilution buffer, and secondary antibody dilution buffer were from Beyotime Institute of Biotechnology (Haimen, China). Trypsin was from Promega (Madison, USA). Nonlinear immobilized pH gradient (IPG) strips were purchased from GE (Piscataway, USA). Chemicals used for 2DE were purchased from Amresco (Solon, USA). ZnSO4∙7H2O was purchased from Aladdin (Shanghai, China). All water used in experiments was Millipore Milli-Q filtered at a resistivity greater than or equal to 18.25 MΩ·cm. Culture dishes, and polyvinylidene difluoride (PVDF) membranes were from Millipore (Bedford, USA). Primary antibodies were purchased from the following vendors: anti-actin antibody (AA128, Beyotime); Hsp90α (D1A7) rabbit mAb (CST#8165) and hnRNPA1 (D21H11) rabbit mAb (CST#8443) (both Cell Signaling Technology). Secondary antibodies, including goat anti-rabbit lgG-HRP (sc-2004) and goat anti-mouse lgG-HRP (sc-2005), were purchased from Santa Cruz Biotechnology.
6
Fluorescence Microscopy Cell Staining Protocol
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For fluorescence microscopy procedures, cells were seeded on circular glass coverslips (WHB Scientific, #WHB-12-CS-LC) placed in 12-well plates. Cells, transfected with expression vectors for (fluorescent)-tagged proteins or used in OMV endocytosis or gentamicin protection assays, were washed with PBS and fixed with 4% paraformaldehyde (Biosharp, #BL539A) for 20 min at room temperature. Cells were subsequently incubated with QuickBlock blocking and permeabilization buffer (Beyotime, #P0260) for 40 min before staining for 12 h at 4 °C with primary antibodies diluted in primary antibody dilution buffer (Beyotime, #P0262). Finally, slides were incubated for 2 h at 25 °C with secondary antibodies diluted in secondary antibody dilution buffer (Beyotime, #P0265). For nuclear staining, 10 μg/mL DAPI solution (Solarbio, #C0065) was used for 15 min at room temperature. Images were acquired on Zeiss LSM880 or Olympus FV3000 microscopes.
7
Bovine Preadipocyte Protein Extraction and Western Blot
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Protein was obtained from bovine preadipocytes using a protein extraction kit, supplemented with PMSF (Beyotime, Shanghai, China), and protein concentration was quantified with the Quantitative Protein Kit (the BCA method) (APPLYGEN, Beijing, China). For Western blot, 20 μg total cellular protein was separated on a 10% polyacrylamide gel, and then transferred onto PVDF. After blocking in Quick Block™ Western blocking solution (Beyotime, Shanghai, China) for 30 min at RT, the PVDF was sequentially incubated overnight at 4 °C with primary antibodies, respectively. Table 1 lists the dilution factor and company name of different antibodies. After washing in TBST-1× buffer 3 times (10 min each time) (Sangon Biotech Co., Ltd., Shanghai, China), the Goat anti-rabbit HRP antibody (absin, 1:10,000) diluted in Secondary Antibody Dilution Buffer (Beyotime, Shanghai, China) was incubated for 2 h, and washed in TBST-1× buffer.
8
Immunofluorescence Staining of Osteocalcin in Bone
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Immunofluorescence staining was performed as previously described (32) on 5-μm paraffin sections of the decalcified tibiae. Briefly, the sections were dewaxed with xylene and rehydrated with 100% ethyl alcohol, 95% ethyl alcohol, 70% ethyl alcohol, and PBS. Sections were then performed antigen retrieval with citrate buffer (pH6.8) and incubated with immunostaining permeabilization solution with saponin (Beyotime, cat# P0095) for 10 min, and blocked with immunol staining blocking buffer (Beyotime, cat# P0102) for 1 h at room temperature. Following that, the Ocn (Bioss Antibodies, cat# BS-4917R) antibody was diluted into the primary antibody dilution buffer (Beyotime, cat# P0262) with 1:300 ratio, and the sections were incubated with the diluted primary antibody for overnight at 4 °C. Sections were washed with 1x PBS for 3 times and incubated with goat anti-rabbit alexa fluor 488 secondary antibody (Invitrogen, cat# A11008) diluted by secondary antibody dilution buffer (Beyotime, cat# P0265) at room temperature for 1 h. The sections were washed with 1 × PBS for 3 times and immediately mounted with mounting medium with DAPI (Abcam, cat# ab104139). The Ocn signal was acquired with Nikon Confocal A1R system under 20x objective lens.
9
Alzheimer's Disease Pathogenesis Examination
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Aβ(1-42) was obtained from Sigma-Aldrich (St. Louis, MO, United States). Advanced RPMI 1640 medium (Gibco), fetal bovine serum (FBS), and RT-PCR kit were purchased from TransGen Biotech Limited Company (Beijing, China). Penicillin–streptomycin solution (100X), trypsin–EDTA solution, SDS–PAGE sample-loading buffer (5X), cell counting kit-8, primary antibody dilution buffer, secondary antibody dilution buffer, phenylmethanesulfonyl fluoride (PMSF), RIPA lysis buffer, and β-actin mouse monoclonal antibody were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Fast Wright-Giemsa’s stain was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The primary antibodies such as NF-κB, IκBα, p-IκBα, Bax, Bcl-2, IL-1β, TNF-α, COX-2, and iNOS were purchased from Cell Signaling Technology, Inc. (Beverly, MA, United States). Caspase-3, cleaved-PARP and PARP antibodies were obtained from abcam (Cambridge, MA, United States). Primers used for RT-PCR analysis were synthesized by Invitrogen (Minato, Tokyo, Japan).
10
Signaling Protein Quantification Protocol
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Biological materials and reagents were acquired as follows: antibodies against Src, phospho-Src (Tyr416), phospho-Akt (Thr308), Akt, Syk, phospho-Syk (Tyr525/526), GSK-3β, phospho-GSK-3β (Ser9), PLCγ2, phospho-JNK (Thr183/Tyr185), JNK, p38 MAPK, and phospho-p38 MAPK (Thr180/Tyr182) were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Phospho-PLCγ2 (Tyr 759) was acquired from GeneTex (Irvine, California, US). Peroxidase-labeled antibodies to rabbit IgG were obtained from SeraCare (Milford, MA, USA); β-actin was acquired from GeneTex (Irvine, CA, USA); PageRuler Prestained Protein Ladder from ThermoFisher Scientific (Shanghai, China); 5× SDS-PAGE Sample Loading Buffer and BCA Protein Assay Kit from Biosharp (Hefei, Anhui, China); polyvinylidene difluoride (PVDF) membranes from MilliporeSigma (Burlington, MA, USA); and electrochemiluminescence (ECL) western blotting detection reagent, SDS-PAGE Gel Preparation Kit, Primary Antibody Dilution Buffer, Secondary Antibody Dilution Buffer, and Western Blocking Buffer from Beyotime Biotechnology (Shanghai, China).
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