Cells were cross-linked in 1% (vol/vol) formaldehyde for 10 minutes at room temperature. Then, 0.125 M glycine was used to quench the cross-linking reaction. Cells were pelleted and washed with ice-cold phosphate-buffered saline. Next, cell nuclei were isolated and a Bioruptor sonicator (Diagenode) was used to shear chromatin DNA. An amount of 5 μL of p53 antibody (1:2,000, Cat No: ab28, Abcam) diluted in phosphate-buffered saline buffer was added to Dynabeads Protein A (Invitrogen) beads and incubated for 3 hours at 4°C with rotation. An amount of 5 μL of normal rabbit immunoglobulin G (Cell Signaling, CA, USA) was used as negative control. Then, the dynabeads-antibody complexes were incubated with sheared chromatin DNA overnight at 4°C. After immunoprecipitation, the precipitated complex was treated with RNase A and Proteinase K, and incubated at 65°C overnight to reverse cross-links. Primers were designed and quantitative RT-PCR (qRT-PCR) was performed. The sequence of primers for HOTAIR promoter (110 bp) was as follows: F 5′-GCCCTTCTCCTAGCCCACCG-3′, R 5′-GTGGGGACCCGCTAGACCTG-3′; sequence of primers for p53 promoter (125 bp) was as follows: F 5′-CAAAGAAATGGAGCCGTGTA-3′, R 5′-GCCTCCTAAAGTGCCAAGAT-3′. Chromatin immunoprecipitation (ChIP)-qPCR data were normalized either by the percent input method or relative to the immunoglobulin G control.
Normal rabbit immunoglobulin g
Manufactured by Cell Signaling Technology
Sourced in United States
Normal rabbit immunoglobulin G is a purified preparation of immunoglobulins from normal rabbit serum. It serves as a control for experiments involving the use of rabbit-derived antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
Bioruptor sonicator, by Diagenode (1 mentions)
Ab4729, by Abcam (1 mentions)
Simplechip plus sonication chromatin ip kit, by Cell Signaling Technology (1 mentions)
Sc 285, by Santa Cruz Biotechnology (1 mentions)
Protein a beads, by Cytiva (1 mentions)
Chip assay kit, by Beyotime (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti gapdh sc 47724, by Santa Cruz Biotechnology (1 mentions)
Anti myc sc 40, by Santa Cruz Biotechnology (1 mentions)
Anti egln1, by Cell Signaling Technology (1 mentions)
Anti hydroxy hif1α pro564, by Cell Signaling Technology (1 mentions)
Anti set7, by Cell Signaling Technology (1 mentions)
Anti set7, by Abcam (1 mentions)
Mg132, by Merck Group (1 mentions)
Anti β actin, by ABclonal (1 mentions)
Anti hif 1α, by Cell Signaling Technology (1 mentions)
Anti ha, by Fortrea (1 mentions)
E cadherin, by Merck Group (1 mentions)
Hsp70, by Enzo Life Sciences (1 mentions)
11 protocols using normal rabbit immunoglobulin g
1
ChIP-qPCR for p53 and HOTAIR
2
Histone H3K27ac ChIP-qPCR Protocol
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According to the manufacturer's instructions, chromatin immunoprecipitation (ChIP) assays were conducted using a Simple ChIP Plus Sonication Chromatin IP Kit (Cell Signaling Technology). HPAEpiC cells were fixed with 1% formaldehyde at 37°C for 10 min, incubated in a lysis buffer and sonicated to shear genomic DNA. Soluble chromatin was incubated with an anti‐H3K27ac antibody (ab4729, Abcam) and normal rabbit immunoglobulin G (2729, Cell Signaling Technology) at 4°C for 12 h. The immunoprecipitate was bound to protein G magnetic beads, and DNA–protein crosslinking was terminated by incubation at 65°C for 2 h. Finally, the immunoprecipitated DNA sequences were quantified by PCR (Table S4 ).
3
ErbB Receptor Activation and Phosphorylation
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CHO cells expressing ErbB constructs were serum starved for up to 4 h. CHO ErbB3wt-mCitrine cells were stimulated with 12 nM HRG for 2 min. All cells were washed with ice-cold PBS on ice and lysed with cold NP-40 lysis buffer (Yang et al., 2007 (link)). Protein concentrations in cleared lysates were measured by BCA assay (Pierce, Rockford, IL). Supernatants were precleared with Protein A beads (Amersham GE Healthcare, Chicago, IL) and normal rabbit immunoglobulin G (Cell Signaling) followed by overnight incubation at 4°C with primary antibodies against ErbB2 (RB9040; Neomarkers) or ErbB3 (sc285 [Santa Cruz Biotechnology] or ErbB3-XP [Cell Signaling]). Proteins in immune complexes were either resuspended in reaction buffer for the in vitro kinase assay or denatured and separated by SDS–PAGE, transferred to nitrocellulose, and probed with the pan-PY antibody anti-PY20-HRP (Santa Cruz Biotechnology) to detect all phosphorylated proteins in the immunoprecipitate or with primary antibodies against ErbB2 or ErbB3 and HRP-conjugated secondary antibodies. Blots were revealed and detected as described.
4
ChIP Assay for SOX4 Transcription Factor
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Chromatin immunoprecipitation (ChIP) assay was performed according to the protocol of ChIP Assay Kit (Beyotime, Nanjing, China). In brief, cells were cross-linked with 37% formaldehyde for 10 min at room temperature. The reaction was stopped by 125 mM glycine for 5 min at room temperature. The cells were scraped and lysed in the SDS lysis buffer containing 1 mM PMSF. The lysate was sonicated and centrifugated. Then, the supernatant was immunoprecipitated overnight at 4°C with antibody anti-SOX4 (1:100, Cell Signaling Technology, Danvers, MA, USA). An equal mass of Normal Rabbit immunoglobulin G (1:1,000, Cell Signaling Technology, USA) was used as negative control. Following immunoprecipitation, the supernatant was transferred to protein A/G-agarose beads and rotated at 4°C for 45 min. The DNA was collected from beads and used as the template for real-time PCR using PrimerChIP (Table 1 ). The relative quantity of DNA was determined by comparing the sample fluorescence to the fluorescence values measured from the total chromatin input dilution series.
5
Antibody Procurement and Utilization
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Antibodies including anti-EGLN1 (#4835), anti-HIF1α (#36169), anti-hydroxy-HIF1α (Pro564) (#3434), anti-SET7 (#2825), and normal rabbit immunoglobulin G (#2729) were purchased from Cell Signaling Technology. Anti-β-actin (#AC026) was purchased from ABclonal. Anti-Flag (#F1804) was purchased from Sigma. Anti-HA (#901515) was purchased from Covance. Anti-Myc (#SC-40) and Anti-GAPDH (#SC-47724) antibody was purchased from Santa Cruz Biotechnology. anti-SET7 (#ab124708) was purchased from Abcam. MG-132 (#474790) was purchased from Sigma. Cycloheximide (#HY-12320) was purchased from MCE.
6
Comprehensive Antibody Resource for Cell Biology
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Anti-PAR6β (BC31AP), anti-Lgl2 (N13AP), and anti-Lgl2-S653P antibodies have been described previously (Yamanaka et al., 2003 (link)). Anti-GP135 (3F2/D8) was a kind gift from George K. Ojakian (State University of New York, Brooklyn, NY). Other antibodies were purchased as follows: Lgl2 (Abnova, Taipei, Taiwan), Lgl1 (Sigma-Aldrich, St. Louis, MO), VprBP (Proteintech Group, Chicago, IL), DDB1 (Bethyl, Montgomery, TX), Cul4A (Bethyl), Cul4B (Proteintech Group), Cdt2 (Novus Biologicals, Littleton, CO), PKC iota (BD, Franklin Lakes, NJ), zonula occludens-1 (ZO-1; Santa Cruz Biotechnology, Dallas, TX), p27kip1 (BD), p16INK4a (Cell Signaling, Danvers, MA), p21Waf1/Cip1 (Santa Cruz Biotechnology), Skp2 (Santa Cruz Biotechnology), Cdh1 (Abcam, Cambridge, United Kingdom), cyclin A (Santa Cruz Biotechnology), cyclin B1 (Santa Cruz Biotechnology), HSP70 (Enzo Life Sciences, Farmingdale NY), EDD1 (Bethyl), glyceraldehyde-3-phosphate dehydrogenase (Abcam), β-Actin (Sigma-Aldrich), E-cadherin (Sigma-Aldrich), V5 (Invitrogen), HA (Roche, Basel, Switzerland), SBP (Santa Cruz Biotechnology), Myc (Millipore, Billerica, MA; Cell Signaling), BrdU (BD; Abcam), and normal rabbit immunoglobulin G (Cell Signaling).
7
Immunoprecipitation Analysis of CRY1 and CRY2 Complexes
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For immunoprecipitation analysis (fig. S9B), the U2OS cell lysates were harvested in radioimmunoprecipitation assay buffer (9806, Cell Signaling) with protease inhibitor cocktail (11697498001, Sigma-Aldrich, St. Louis, MO, USA). Protein A–coated magnetic beads (10006D, Thermo Fisher Scientific, Waltham, MA, USA) were preincubated with 2 μg of CRY1 (A302-614A, Bethyl Laboratories), CRY2 (A302-615A, Bethyl Laboratories), and normal rabbit immunoglobulin G (2729, Cell Signaling) at 4°C for 6 hours. The antibody-conjugated beads were incubated with lysate containing equal amounts of total protein at 4°C overnight. The final immune complexes were analyzed by immunoblot assay using anti-HSP90AA1 (HSP90 α) (SMC-108D, StressMarq Biosciences, Victoria BC V8N 4G0, Canada), anti-HSP90AB1 (HSP90 β) (37-9400, Thermo Fisher Scientific), CRY1 (A302-614A, Bethyl Laboratories), CRY2 (A302-615A, Bethyl Laboratories), and anti-actin (4967S, Cell Signaling).
8
Immunoprecipitation and Ubiquitin Assay
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SH-SY5Y cells were treated as described in Section 2.1 . Cells were lysed in EBC buffer and centrifuged, and then the soluble supernatant was collected. Next, we cleared the supernatant with protein A-sepharose and added anti-ZNF746 antibody or normal rabbit immunoglobulin G (Cell Signaling Technology) to the supernatant to incubate overnight at 4 °C. The following day, the supernatant was added with A-sepharose beads (0.1 g/L) and reacted for 4 h at 4 °C. Finally, the beads were washed with EBC buffer and the phosphate buffer. Immunoprecipitation complexes were obtained by centrifugation (14,600× g) for 10 min at 4 °C and then assayed for ubiquitin level by Western blotting.
9
Co-immunoprecipitation of PR50 and NOVA1
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First, PR50 DNA was subcloned into the pCMV-HA vector (Clontech), and NOVA1-coding cDNA was subcloned into the pCMV-Myc vector (Clontech). The plasmids were then co-transfected into 293T cells using Lipofectamine 2000 (Invitrogen). After 24 h, the cells were lysed and supernatants were collected. For co-immunoprecipitation, we first purified the supernatant with Protein G-Sepharose beads and then immunoprecipitated it with rabbit anti-HA tag antibody (Cell Signaling Technology) or normal rabbit immunoglobulin G (Cell Signaling Technology) at 4 °C for 2 h, followed by incubation of Protein G-Sepharose beads for 1 h. Finally, the immunoprecipitated complexes were washed and subjected to Western blotting. NOVA1 and PR50 proteins were detected using mouse anti-Myc and mouse anti-HA tag antibodies (Cell Signaling Technology), respectively. In addition, the vectors of PR50 and NOVA1 were exchanged to perform the above experiments.
10
ChIP Profiling of HDAC1 and Acetylated Histone H3
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ChIP experiments were performed on coronary artery tissue sections, which were prepared as described above, using rabbit anti-human HDAC1 (cat. no. 34589) and rabbit anti-histone H3 acetyl (cat. no. 9927) primary antibodies (both 1:100; Cell Signaling Technology, Inc.), and normal rabbit immunoglobulin G (cat. no. 12-370; Upstate Biotechnology; Merck Millipore, Billerica, MA, USA) as a negative control. All steps were carried out as previously described (11 (link)). In brief, the cells were fixed with 1% formaldehyde (Sigma-Aldrich; Merck KGaA) for 30 min at 37°C, then quenched with 125 mM glycine for 10 min at room temperature to form DNA-protein cross-links. Samples were sonicated on ice until chromatin fragments were 200–1,000 bp in size, then incubated with antibodies at 4°C overnight. PCR amplification was performed under the following conditions: Thirty-three cycles in total, consisting of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 30 sec.
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