ChIP was performed similarly to described methods (Renthal et al., 2008 (link); Tsankova et al., 2004 (link)). Hippocampi were subdissected and fixed in 1% formaldehyde. Samples were sonicated to generate genomic fragments 200–1000 bp in length and pre-cleared with Protein A beads (Millipore) prior to incubation with antibodies listed in Supplemental Table 1 at 4°C overnight. The antibody-chromatin complex was immunoprecipitated with Protein A beads and then washed with a series of buffers (Millipore), then chromatin was then eluted and reverse cross-linking was performed with Proteinase K. DNA was purified via phenol-chloroform extraction. Final DNA concentration was measured using the NanoDrop 2000. Quantitative PCR was performed with an ABI 7500 PCR machine using SYBR green as a fluorophore. Primers used to amplify the c-fos promoter and β-actin promoter are listed in Supplemental Table S2 .
Protein a beads
Manufactured by Merck Group
Sourced in United States, Germany
Protein A beads are a type of affinity chromatography resin used for the purification of antibodies. These beads are made of agarose or polymer matrices and have protein A immobilized on their surface. Protein A is a bacterial protein that binds specifically to the Fc region of immunoglobulins, particularly IgG. The beads can be used to capture and purify antibodies from complex mixtures, such as cell culture supernatants or serum samples.
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Lab products found in correlation
Pcr purification kit, by Zymo Research (2 mentions)
Anti gfp antibody, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Chloroquine, by Merck Group (2 mentions)
Anti tubulin antibody, by Merck Group (2 mentions)
Anti myc, by Santa Cruz Biotechnology (2 mentions)
Antibodies against sgk1 af3200, by R&D Systems (2 mentions)
Antibodies against phospho akt substrate rxrxxs t 23c8d2, by Cell Signaling Technology (2 mentions)
Fluorescein and rhodamine conjugated secondary antibodies, by Jackson ImmunoResearch (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
Mg132, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Glutathione beads, by Merck Group (2 mentions)
Zymo spin chip kit, by Zymo Research (2 mentions)
Rabbit igg isotype control, by Merck Group (2 mentions)
Dna isolation kit, by Corning (2 mentions)
Sirt1 antibody, by Merck Group (1 mentions)
Sensimix sybr fluorescein kit, by Meridian Bioscience (1 mentions)
62 protocols using protein a beads
1
Chromatin Immunoprecipitation in Hippocampus
2
ChIP-qPCR Profiling of ANAC055 Regulation
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For the ChIP assay, 21-d-old Col-0, cau1, and 35S:EYFP-CAU1/cau1 plants grown under long-day conditions were harvested. Approximately 4 g of plant material was cross-linked for 20 min in 1% formaldehyde. ChIP assays were performed as previously described (Vartanian et al., 1994 (link); Ascenzi and Gantt, 1999 (link)). The sonicated chromatin extractions were immunoprecipitated overnight with antisymmetric dimethyl-H4R3 antibody (Abcam) for plants of Col-0 and cau1, with an anti-GFP (green fluorescent protein) antibody (Invitrogen) for plants of 35S:EYFP-CAU1/cau1, or without antibody. Incubation of chromatin with mouse IgG (Abcam) served as a mock immunoprecipitation control. Protein A beads (Millipore) were used to capture the immunocomplexes. After reverse cross-linking and proteinase-K digestion, the DNA was extracted with phenol-chloroform and then precipitated with ethanol. The immunoprecipitated DNA was subsequently used for qPCR. The sequences were amplified from –1388 to 646 bp of the ANAC055 gene and each DNA fragment was approximately 120 bp in length. Primers used for ChIP-qPCR were as follows: Region A (ANAC055-1), region B (ANAC055-2), region C (ANAC055-3), region D (ANAC055-8), region E (ANAC055-9), region F (ANAC055-10), region G (ANAC055-11), and the primer sequences are given in Table S1 at Dryad. TUB8 was used as a control (Mathieu et al., 2005 (link)).
3
SIRT1 Immunoprecipitation Protocol
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Sirt1 over-expressing and control C3HT101/2 cells were lysed in lysis buffer (50mM TRIS-HCl, pH 7.4/1% NP-40, 0.25% Sodium deoxycholate/150 mM NaCl, 1mM EDTA/1mM PMSF supplemented with protease inhibitor and phosphatase inhibitor). Marrow from tibiae and femora was flushed with the same buffer, then cleared by centrifugation at 12,000 RPM for 15 min at 4°C. Immunoprecipitation was carried out by preclearing 1 μg of protein in 300 μl lysis buffer with 30 μl protein A beads (Millipore) for 1 h and incubating the lysates with 4 μl of the SIRT1 antibody (Millipore, 07-131), rotating over night at 4°C. For precipitation, 30 μl protein A agarose beads were added followed by incubation at 4°C for 3 h. Immunoprecipitates were washed extensively and eluted twice with x2 Laemmli buffer. Proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore).
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Sirt1 over-expressing and control C3HT101/2 cells were lysed in lysis buffer (50mM TRIS-HCl, pH 7.4/1% NP-40, 0.25% Sodium deoxycholate/150 mM NaCl, 1mM EDTA/1mM PMSF supplemented with protease inhibitor and phosphatase inhibitor). Marrow from tibiae and femora was flushed with the same buffer, then cleared by centrifugation at 12,000 RPM for 15 min at 4°C. Immunoprecipitation was carried out by preclearing 1 μg of protein in 300 μl lysis buffer with 30 μl protein A beads (Millipore) for 1 h and incubating the lysates with 4 μl of the SIRT1 antibody (Millipore, 07-131), rotating over night at 4°C. For precipitation, 30 μl protein A agarose beads were added followed by incubation at 4°C for 3 h. Immunoprecipitates were washed extensively and eluted twice with x2 Laemmli buffer. Proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore).
4
Co-Immunoprecipitation Protocol for Protein Interactions
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Co-immunoprcipitation in cells was described previously22 (link). Simply, cells or tissues were lysed in buffer A (1% NP-40, 10% glycerol, 135 mM NaCl, 20 mM Tris, pH 8.0, supplemented with protein inhibitors). Lysates were added with 20 μl anti-Flag beads or 4 μg indicated antibody or control IgG and incubated overnight. For antibodies, Protein-A beads (Millipore) were added 2 h before washing with buffer A for four times.
5
Immunoprecipitation of Protein Complexes
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After washed twice with cold PBS, cultured cells were collected and resuspended in RIP buffer (200 mM Tris-HCl, pH 7.5; 20 mM MgCl2; 300 mM NaCl; 10% glycerol; 0.5% NP-40; 0.5% Triton-X 100) plus protease inhibitors and phosphatase inhibitors. Then cell suspensions were incubated on ice for 10 min and lysed by sonicator. After centrifugation at 14,000 × g for 10 min at 4 °C, cell lysates were incubated with primary antibodies or control IgG overnight at 4 °C, and further with protein A beads (Millipore, pre-treated with 5% bovine serum albumin in RIP buffer) for another 4 h. The beads were washed three times and boiled for 5 min in SDS-PAGE sample buffers to elute bound proteins for Western blotting.
6
ChIP Assay for ZEB1 Binding on DDR1 Promoter
7
Chromatin Immunoprecipitation of REST in Pediatric Brain Cancer Cells
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Actively dividing SU-DIPG (IV, VII, and XIII) cells were fixed with 1% formaldehyde. Cell pellets (5 million per reaction) were suspended in lysis buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0), 1% SDS, protease inhibitors (Sigma) and chromatin sonicated with a Bioruptor Pico (Diagenode). 1% of the recovered material was saved as input and the remainder diluted fivefold in ChIP dilution buffer (16.7 mM Tris-HCl (pH 8.0), 167 mM NaCl, 1.2 mM EDTA (pH 8.0), 1.1% Triton X-100, protease inhibitors) and precleared. REST (Millipore, 07-579) or IgG (Santa Cruz, sc2027) antibodies were added overnight before incubation with protein-A beads (Millipore) and washing once each with low-salt, high-salt, and lithium chloride immune complex buffers, and twice with TE buffer. DNA was eluted with 1% SDS and 0.1 M NaHCO3 and cross-links reversed by incubation with NaCl for 4 h at 65°. DNA was purified with PCR purification kit (Zymo Research) and analyzed by qPCR with a 2× SensiMix SYBR & Fluorescein Kit (Bioline) on a Lightcycler 96 machine (Roche).
8
ChIP Analysis of GL2 Transcription Factor
9
Bispecific Antibody Production in 293S Cells
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293S cells were transfected using DNA constructs of bispecific antibodies and NanoFect transfection agent and incubated in Freestyle F17 medium with 8 mM Glutamine and 0.1% Pluoronic F68 surfactant in suspension bottles using shaker at 37 °C and 5% CO2. The supernatant was collected at days 3–7 for antibody purification by centrifugation for 12 min at 3000× g. The filtered supernatant was used for the isolation of antibody using column with protein A beads (Millipore, Burlington, MA, USA). After washing column with 1× PBS, the antibody was eluted with Pierce IgG elution buffer (ThermoFisher). The concentration of bispecific antibody was estimated with Nanodrop instrument and BCA protein assay kit (Thermo Fisher). The purified antibodies were run on SDS gel and used for functional analyses.
10
Purification of Brg1 from Differentiated C2C12 Cells
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