ChIP was performed on total thymocytes from WT or HDAC3-cKO using the SimpleChIP Enzymatic Chromatin IP kit, (Cell Signaling, #9002) per manufacturer’s guidelines. Chromatin was incubated with α-acetyl Histone 3 antibody (Millipore) and control antibodies (Cell Signaling, #9002) overnight at 4°C with rotation. Extracted DNA was analyzed by real-time PCR using previously published primer sequences spanning the RORC promoter (26 (link)).
Control antibodies
Manufactured by Cell Signaling Technology
Control antibodies are essential laboratory tools used to validate the specificity and performance of primary antibodies in various immunoassays. They provide a reliable reference point to ensure the accuracy and consistency of experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Simplechip enzymatic chromatin ip kit, by Cell Signaling Technology (2 mentions)
Micrococcal nuclease, by Cell Signaling Technology (1 mentions)
Anti stat1 antibody, by Cell Signaling Technology (1 mentions)
Bioruptor, by Diagenode (1 mentions)
Ez chip kit, by Merck Group (1 mentions)
Bioruptor ucd 200, by Diagenode (1 mentions)
3 protocols using control antibodies
1
ChIP-PCR Analysis of RORC Promoter
2
STAT1 Chromatin Immunoprecipitation in BMDMs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIP assay was performed with SimpleChIP® Enzymatic Chromatin IP Kit (Cell Signaling Technology). In brief, BMDMs treated with 100 U/ml IFN‐β for 4 h were fixed in 1% formaldehyde for 10 min. Chromatin was fragmented by using micrococcal nuclease (Cell Signaling Technology), and the cell membrane was broken down by Bioruptor (Diagenode). 2% of samples were aliquoted for input controls, and the remaining samples were incubated with anti‐STAT1 antibody (Cell Signaling Technology) and control antibodies (Cell signaling Technology) overnight at 4°C with rotation. The precipitated DNA was purified and conducted qPCR assay by using iQ SYBR Green Supermix with the primers listed in the Reagents and Tools Table. The data were analyzed by the following formula: % of input = (input dilution factor/bound dilution factor) × 2 (input CT − bound CT) × 100 and then normalized to IgG control as 1.
3
ChIP Assay for FLAG-tagged Protein Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromatin immunoprecipitation (ChIP) experiments were performed mostly according to the manufacturer's instructions (EZ-ChIP™ kits, Millipore, USA). Briefly, for each ChIP assay, 5 × 106 cells expressing MITR-FLAG were employed. Chromatin was cross-linked with 1% formaldehyde (Sigma, USA) and sheared using a Bioruptor UCD-200 (Diagenode, Belgium), with pulses of 30 s on and 30 s off for 30 min. The samples were then immunoprecipitated overnight with rabbit monoclonal anti-FLAG (Cell Signaling, 14793, 10 μg), or the same amount of control antibodies (Cell Signaling, 10 μg). The next day, the cross-links in the protein-antibody-DNA complexes were removed by proteinase K to obtain the FLAG-associated and negative control DNA samples. For the ChIP-qPCR assay, the two DNA samples were used as the DNA template, and primers targeting different regions of the IL11 promoter were designed (Table S1
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!