AGO2 binding RNA immunoprecipitation analysis were performed as described (14 (link)). MM1S V-GFP and MM1S V-miR-221-222-GFP stable transduced cells (3×107 per RIP experiment) were washed with ice-cold phosphate buffer solution (PBS) and dispensed into 1 ml of cell lysis buffer (50 mM Tris-HCl PH 7.5, 150 mM NaCl,1 mM EDTA, 1% NP 40) containing Protease inhibitor (#5871S, Cell Signaling) and RNase inhibitor (#N808-0119, Invitrogen) for 20 min on ice. Lysates were microcentrifuged at maximum speed for 20 min at 4°C, and supernatants were collected and pre-cleared with 20 μl of Protein A and G Dynabeads coupled with 5 μg of normal Rabbit IgG (#SC-3888, Santa Cruz). The beads were removed by centrifugation and the supernatants incubated with Dynabeads A and G coupled with 5 μg of anti-AGO2-specific antibody (#2897S, Cell Signaling) or 5 μg IgG isotype control antibody (#SC-3888, Santa Cruz) overnight at 4°C, followed by three time washes with washing buffer (50 mM Tris-HCL Ph 7.5, 300 mM NaCl, 1mM EDTA, 1% NP 40, Protease inhibitor). After pulldown by centrifugation, RNA was extracted using 1000 μl TRIZOL reagent (Invitrogen). Quantitative RT-PCR detection of pulled-down PUMA mRNA and miR-221/222 was performed as described in the section of Q-RT-PCR.
Sc 3888
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-3888 is a laboratory instrument designed for sample preparation and analysis. This equipment is used for the isolation, purification, and characterization of biological molecules, such as proteins, nucleic acids, and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Multicycle av software, by Phoenix Pharmaceuticals (2 mentions)
Rnase a, by Thermo Fisher Scientific (2 mentions)
Propidium iodide, by Thermo Fisher Scientific (2 mentions)
Cytomics fc500 flow cytometer, by Beckman Coulter (2 mentions)
Sc 2025, by Santa Cruz Biotechnology (2 mentions)
Protease inhibitor, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Santa Cruz Biotechnology (1 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Fraction 5, by Merck Group (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Sc 9081, by Santa Cruz Biotechnology (1 mentions)
A21441, by Thermo Fisher Scientific (1 mentions)
Isotype rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Anti his, by Proteintech (1 mentions)
Anti ha, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Leica tcs sp8, by Leica Microsystems (1 mentions)
10 protocols using sc 3888
1
AGO2-Bound RNA Immunoprecipitation in MM1S Cells
2
Cell Cycle and Oct4 Expression Analysis
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The cellular probes were analyzed using a Cytomics FC500 flow cytometer (Beckman Coulter). The cell suspensions (106/ml) were prepared using the 0.05% trypsin-EDTA (HyClone) treatment. To analyze the cell cycle distribution, the cells were fixed with cold 70% ethanol. After fixation and triple washing with PBS the cells were incubated in PBS containing 20 μg/ml of propidium iodide (Invitrogen/Molecular Probes) and 200 μg/ml of RNAse A (Fermentas) for 30 min. After staining, the probes were analyzed immediately. The histograms were analyzed using the MultiCycle AV Software (Phoenix Flow Systems, USA).
For the flow cytometry analysis of Oct4-expressing cells, the suspensions of cells (106/ml) were fixed with 3% paraformaldehyde in PBS for 15 min, washed with PBS, and treated with 0.5% Triton X-100, 3% bovine serum albumin, Fraction V (Sigma), and rabbit anti-Oct4 antibodies (1 : 200, Santa Cruz Biotechnology) in PBS for 40 min. After washing, the cells were incubated in a PBS solution containing 0.5% Triton X-100, 3% bovine serum albumin, and secondary chicken anti-rabbit antibodies conjugated with Alexa-488 (1 : 1000, Molecular Probes) for 30 min. For the negative control, the cells were treated with normal rabbit IgG (sc-3888, Santa Cruz Biotechnology) and then with the same secondary antibody solution described above.
For the flow cytometry analysis of Oct4-expressing cells, the suspensions of cells (106/ml) were fixed with 3% paraformaldehyde in PBS for 15 min, washed with PBS, and treated with 0.5% Triton X-100, 3% bovine serum albumin, Fraction V (Sigma), and rabbit anti-Oct4 antibodies (1 : 200, Santa Cruz Biotechnology) in PBS for 40 min. After washing, the cells were incubated in a PBS solution containing 0.5% Triton X-100, 3% bovine serum albumin, and secondary chicken anti-rabbit antibodies conjugated with Alexa-488 (1 : 1000, Molecular Probes) for 30 min. For the negative control, the cells were treated with normal rabbit IgG (sc-3888, Santa Cruz Biotechnology) and then with the same secondary antibody solution described above.
3
Cell Cycle and Oct4 Expression Analysis
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To analyze the cell cycle distributions, the cells were suspended in 0.05% Trypsin-EDTA (HyClone) and fixed with cold 70% ethanol. After triple washing with PBS the cells (106/ml) were incubated in PBS containing 20 μg/mL of propidium iodide (Molecular Probes) and 200 μg/mL of RNAse A (EN0531, Fermentas, Lithuania) for 30 min. The probes were analyzed immediately after staining using a Cytomics FC500 flow cytometer (Beckman Coulter, USA) and MultiCycle AV Software (Phoenix Flow Systems, San Diego, CA, USA).
For the flow cytometry analysis of Oct4-expressing cells, the cells (106/ml) were fixed with 3% paraformaldehyde in PBS for 15 min, washed with PBS and treated with 0.5% Triton X-100, 3% BSA and rabbit anti-Oct4 antibodies (1:200, sc-9081, Santa Cruz Biotechnology) in PBS for 40 min. After washing, the cells were incubated in PBS solution with 0.5% Triton X-100, 3% BSA and secondary chicken anti-rabbit antibodies conjugated with Alexa-488 (1:1000, A-21441, Molecular Probes) for 30 min. For the negative control, the cells were treated with normal rabbit IgG (sc-3888, Santa Cruz Biotechnology) and then with the same secondary antibody solution.
For the flow cytometry analysis of Oct4-expressing cells, the cells (106/ml) were fixed with 3% paraformaldehyde in PBS for 15 min, washed with PBS and treated with 0.5% Triton X-100, 3% BSA and rabbit anti-Oct4 antibodies (1:200, sc-9081, Santa Cruz Biotechnology) in PBS for 40 min. After washing, the cells were incubated in PBS solution with 0.5% Triton X-100, 3% BSA and secondary chicken anti-rabbit antibodies conjugated with Alexa-488 (1:1000, A-21441, Molecular Probes) for 30 min. For the negative control, the cells were treated with normal rabbit IgG (sc-3888, Santa Cruz Biotechnology) and then with the same secondary antibody solution.
4
Co-Immunoprecipitation Protocol for APP
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Co-IP was performed as previously described (18 (link)). The following antibodies were used: anti-HA (Sigma-Aldrich, H6908, 1:200), anti-His (Proteintech, 66005-1-Ig, 1:200), isotype rabbit IgG (Santa Cruz Biotechnology, sc-3888, 1:200), and isotype mouse IgG (Santa Cruz Biotechnology, sc-2025, 1:200). A previously described anti-APP rabbit polyclonal antibody RU-369 (1:200; ref. 42 (link)) was used.
5
Quantification of C3aR Expression in Podocytes and RPTECs
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At the end of the incubations, podocytes and RPTECs were fixed in 2% paraformaldehyde and 4% sucrose, then permeabilized with 0.3% Triton X-100 (Sigma-Aldrich). Nonspecific binding sites were blocked with 2% FBS, 2% BSA and 0.2% bovine gelatin. Cells were incubated with a rabbit anti-C3aR (1:50; LS-C382362, Lifespan BioSciences, Seattle, DC, USA) antibody followed by a Cy3-conjugated secondary antibody (1:80; 711-165-152, Jackson ImmunoResearch Laboratories). Nuclei were counterstained with DAPI (Sigma-Aldrich). Negative controls, obtained by incubating cells with rabbit IgG (sc-3888, Santa Cruz) before Cy3-conjugated secondary antibody, exhibited no unspecific signal. Samples were examined using confocal microscopy (Leica TCS SP8, Leica Microsystems). The quantification of C3aR expression was performed on 10–15 random fields per sample. Specifically, the areas corresponding to the staining were measured in pixel2 using the ImageJ software and normalized for the number of nuclei identified by DAPI staining.
6
Chromatin Immunoprecipitation Protocol
7
Flow Cytometry Analyses of Immune Cells
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The following antibodies/reagents were used for flow cytometry analyses: anti-mouse CD31 (sc-1506-R; Santa Cruz Biotechnology), anti-mouse CD45 (sc-25590; Santa Cruz Biotechnology), and normal rabbit IgG (sc-3888; Santa Cruz Biotechnology), used as a negative control. Stained cells were analyzed by flow cytometry (BD FACSCalibur).
8
Optimization of TRPV1 Antibody Detection
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Three polyclonal rabbit anti-TRPV1 antibodies [SC-20813, purified by proprietary techniques (Santa Cruz Biotechnology, CA, USA), ACC-030, affinity purified on immobilized antigen (Alomone Labs, Jerusalem, Israel), LS-C150735, affinity purified (Lifespan Biosciences, WA, USA), and polyclonal IgG goat anti-rabbit secondary antibody-FITC (Santa Cruz Biotechnology) were initially assessed. Testing of fixation and permeabilization kits [CALTAG™ (Life Technologies); Cytofix/Cytoperm™ (BD Biosciences)], blocking agents [AB serum (from a healthy donor), 10% goat serum (Life Technologies), FcR blocking reagent (Miltenyi Biotechnology, Cologne, Germany) and a mixture of 10% human AB serum, 1% BSA and 0.05% sodium azide, and goat serum mix (10% goat serum + 0.1% BSA + 0.05% sodium azide)] was also performed. Isotype control rabbit IgG, sc-3888 (Santa Cruz Biotechnology) was used as a negative control in a same quantity/dilution as the primary anti-TRPV1 in all experiments. Exclusion of TRPV1 antibody was used as an additional negative control. Protocols were developed to produce the best signal (specific binding) to noise (non-specific binding) ratio for Western blotting and separation of TRPV1 signal from isotype control for flow cytometry.
9
Chromatin Immunoprecipitation of STAT5B in NK Cells
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Splenic NK cells from 10 WT and 13 Stat5 DKI mice were purified (Miltenyi Biotec Inc.), expanded in vitro with 20 ng ml−1 of human recombinant IL-15 (R&D Systems or BioLegend) for 10 days, with fresh rhIL-15 being added every two days, yielding 50–80 million of nearly 100% pure CD3−CD122+NK1.1+ cells. The cells were washed three times with medium, rested in complete RPMI-1640 medium without IL-15 for 2 h, not treated or treated with 40 ng ml−1 of IL-15 for 1 h, and cross-linked with 1% formaldehyde (methanol-free, Pierce, Rockford, IL) at room temperature for 10 min. After sonication, fragmented chromatin equivalent to 10 million cells were immunoprecipitated with control rabbit IgG (sc-3888, Santa Cruz Biotechnology, Dallas TX) or anti-STAT5B (AF1584, R&D Systems) and Magna ChIP Protein A + G Magnetic Beads (16–663, Millipore, Billerica MA). ChIP-Seq DNA libraries were prepared using KAPA LTP Library Preparation Kit (KK8232, Kapa Biosystems, Wilmington, MA) and NEXflex DNA Barcodes-24 (NOVA-514103, BIOO Scientific, Austin, TX), and the libraries were then sequenced on an Illumina HiSeq 3000 platform.
10
ChIP-qPCR protocol for RIP140 binding
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Approximately one hundred and twenty million cells were harvested per experiment. Briefly, protein–DNA complexes were first cross-linked, using 2 mM di(N-succinimidyl)glutarate (DSG), for 50 min on a rotor, then the procedure was performed using High-Sentivity kit (Active Motif, Shanghai) and according to manufacturer’s protocol. Chromatin was sonicated for 30 s on ice followed by 30 s off, for a total of 30 min. Immunoprecipitations were performed with 30 µg of protein–DNA complexes and 4 µg of RIP140 antibody (Ab42126; Abcam) or normal rabbit IgG (sc-3888; Santa Cruz Biotechnology, Dallas, USA). The purified DNAs were amplified with SYBR Green SensiFAST™ SYBR® No-ROX Kit (Bioline, London, UK) by real-time qPCR.
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