Monoclonal rat anti-mouse CD34 FITC-conjugated antibody, monoclonal rat anti-mouse Sca-1 PE-conjugated antibody, polyclonal rat anti-mouse CD11c PE-conjugated antibody, monoclonal rat anti mouse Qa2 antibody, monoclonal rat anti mouse IDO antibody, polyclonal rat anti mouse β-Actin HRP-conjugated antibody, monoclonal chicken anti rat HRP-conjugated antibody (all from Abcam, Cambridge, MA, USA), Dulbecco’s modification of Eagle medium (DMEM), Roswell Park Memorial Institute (RPMI), fetal bovine serum (FBS), enhanced luminol-based chemiluminescent substrate (ECL), Granulocyte-macrophage colony stimulating factor (GM-CSF), Trizol, RIPPA buffer, Bradford kit, protein inhibitor, polyvinylidene difluoride (PVDF) (all from Sigma, Ronkonkoma, NY, USA), cDNA synthesis kit (TAKARA, Japan), PCR SYBR Green kit (TAKARA, Japan), Nycodenz (Axis shield, Norway), Pan DC Microbead MACS (Miltenyibiotec, Germany).
Rippa buffer
Manufactured by Merck Group
Sourced in United States
RIPPA buffer is a laboratory reagent designed for protein extraction and cell lysis. It is a proprietary formulation developed by Merck Group to facilitate the efficient release of proteins from biological samples. The RIPPA buffer aims to provide a consistent and reliable method for protein extraction, supporting various downstream applications in life science research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Protein inhibitor, by Merck Group (1 mentions)
Bradford kit, by Merck Group (1 mentions)
Polyvinylidene difluoride pvdf, by Merck Group (1 mentions)
Cdna synthesis kit, by Takara Bio (1 mentions)
Nycodenz, by Axis-Shield (1 mentions)
Anti gfp, by Abmart (1 mentions)
Anti myc, by Abmart (1 mentions)
Bca protein assay kit, by Merck Group (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Ecl solution, by Cytiva (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Q exactive series mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Anti gfp trap a beads, by Proteintech (1 mentions)
5 protocols using rippa buffer
1
Characterization of mouse myeloid dendritic cells
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Monoclonal rat anti-mouse CD34 FITC-conjugated antibody, monoclonal rat anti-mouse Sca-1 PE-conjugated antibody, polyclonal rat anti-mouse CD11c PE-conjugated antibody, monoclonal rat anti mouse Qa2 antibody, monoclonal rat anti mouse IDO antibody, polyclonal rat anti mouse β-Actin HRP-conjugated antibody, monoclonal chicken anti rat HRP-conjugated antibody (all from Abcam, Cambridge, MA, USA), Dulbecco’s modification of Eagle medium (DMEM), Roswell Park Memorial Institute (RPMI), fetal bovine serum (FBS), enhanced luminol-based chemiluminescent substrate (ECL), Granulocyte-macrophage colony stimulating factor (GM-CSF), Trizol, RIPPA buffer, Bradford kit, protein inhibitor, polyvinylidene difluoride (PVDF) (all from Sigma, Ronkonkoma, NY, USA), cDNA synthesis kit (TAKARA, Japan), PCR SYBR Green kit (TAKARA, Japan), Nycodenz (Axis shield, Norway), Pan DC Microbead MACS (Miltenyibiotec, Germany).
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Monoclonal rat anti-mouse CD34 FITC-conjugated antibody, monoclonal rat anti-mouse Sca-1 PE-conjugated antibody, polyclonal rat anti-mouse CD11c PE-conjugated antibody, monoclonal rat anti mouse Qa2 antibody, monoclonal rat anti mouse IDO antibody, polyclonal rat anti mouse β-Actin HRP-conjugated antibody, monoclonal chicken anti rat HRP-conjugated antibody (all from Abcam, Cambridge, MA, USA), Dulbecco’s modification of Eagle medium (DMEM), Roswell Park Memorial Institute (RPMI), fetal bovine serum (FBS), enhanced luminol-based chemiluminescent substrate (ECL), Granulocyte-macrophage colony stimulating factor (GM-CSF), Trizol, RIPPA buffer, Bradford kit, protein inhibitor, polyvinylidene difluoride (PVDF) (all from Sigma, Ronkonkoma, NY, USA), cDNA synthesis kit (TAKARA, Japan), PCR SYBR Green kit (TAKARA, Japan), Nycodenz (Axis shield, Norway), Pan DC Microbead MACS (Miltenyibiotec, Germany).
2
Protein Interaction Analysis in Arabidopsis
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The cDNA of GmNFYA, GmFVE and GmHDA13 was cloned into pCAMBIA‐2300 vector to generate 35S:GmNFYA‐myc, 35S:GmFVE‐GFP and 35S:HDA13‐myc constructs. Pairwise constructs were transfected into Arabidopsis protoplasts as described previously (Wei et al., 2017 (link)). After incubation at 22 °C for 16 h in darkness, transformed protoplasts were harvested, and total proteins were extracted in Rippa buffer (Sigma). The protein extracts were purified with anti‐myc/anti‐GFP antibody (Sigma/Chromotek) and the immunoprecipitated proteins were separated by SDS–PAGE gel and detected by anti‐myc (Abmart) or anti‐GFP (Abmart) antibodies.
3
Western Blot Protein Analysis Protocol
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The cells were lysed using RIPPA buffer (Sigma-Aldrich; Merck KGaA) and total protein concentration was quantified using the BCA protein assay kit (Sigma-Aldrich; Merck KGaA) following the manufacturer's instructions. Total protein (20 µg) was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to PVDF membrane and blocked with TBS-Tween (0.1%) containing 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) at room temperature for 30 min, and probed with the specific primary antibodies at room temperature for 60 min. Anti-IRS-1 antibody (cat. no. ab52167), anti-Dvl2 antibody (cat. no. ab22616) and anti-β-actin antibody (cat. no. ab8226) were all bought from Abcam, and diluted in 1:2,000 before use. Then, the blot was incubated with specific horseradish peroxidase (HRP)-conjugated secondary antibodies (cat. nos. ab7090 or ab97040; Abcam) at dilution of 1:1,000, and the immunoreactive bands were imaged using enhanced chemiluminescence (Thermo Fisher Scientific, Inc.). The blots were analyzed using ImageJ software (version-2.0; National Institutes of Health).
4
Spinal cord protein expression analysis
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At indicated times after i.a. injection of stimuli (mBSA 100 μg), mice were terminally anesthetized and the ipsilateral spinal cord was collected and homogenized in Rippa® buffer (Sigma–Aldrich, St. Louis, Missouri, USA) containing proteases inhibitors (Complete®- Roche Diagnostics, Indianapolis, USA). The protein concentration of the lysate was determined using Bradford colorimetric assay. The protein samples were separated on SDS/PAGE gels (12% for GFAP and 15% for Iba-1), and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Little Chalfont, UK). The membranes were incubated with primary antibodies against GFAP (1:500; Millipore, Billerica, MA, EUA), Iba-1 (1:200; Wako Chemicals, Osaka, Japan) and β-actin (1:1,000 Millipore, Billerica, Massachusets, EUA) overnight at 4 °C, and then incubated for 3 hours at room temperature with an HRP-conjugated secondary antibodies, anti-mouse (1:3,000) for GFAP, anti-rabbit 1:10,000 for Iba-1 and anti-mouse 1:3,000 for β-actin. The blots were visualized in an ECL solution (Amersham Pharmacia Biotech, Little Chalfont, UK) and exposed in a ChemiDoc MP Imaging System (Bio-Rad Laboratories, Hercules, California, USA).
5
Immunoprecipitation and Mass Spectrometry Analysis of GmNFYA-GFP
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For immunoprecipitation followed by mass spectrometry, soybean transgenic hairy roots harbouring 35S:GmNFYA‐GFP (NFYA‐GFP) were generated and GFP‐overexpressing hairy roots were used as control. Three gram of transgenic hairy roots were harvested and total proteins were extracted in Rippa buffer (Sigma). Anti‐GFP Trap‐A beads (Chromotek) was used for IP. After IP, the proteins were separated by 10% SDSS‐PAGE. The stained protein band was cut and in‐gel digestion was performed by Novogene (Beijing, China). Mass spectrometry measurements were performed on a Q ExactiveTM series mass spectrometer (Thermo Fisher). The resulting spectra were searched against UNIPROT protein database, using soybean as a target organism. Gene ontology (GO) and InterPro (IPR) functional analysis were conducted using the interproscan programme against the non‐redundant protein database (including Pfam, PRINTS, ProDom, SMART, ProSite, PANTHER), and the databases of COG (Clusters of Orthologous Groups) and KEGG (Kyoto Encyclopedia of Genes and Genomes) were used to analyse the protein family and pathway. Proteins uniquely identified in NFYA‐GFP sample but not in control sample were taken into account.
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