The following antibodies were used for ChIP: SRC-3 (Cell Signaling or BD Biosciences), ATF4 (Santa Cruz C-20, and Cat# 11815 Cell Signaling), pSRC-3-S857 (Cell Signaling), and rabbit IgG. ChIP assays were performed according to an EZ ChIP kit (Millipore) with some modification35 (link). Briefly, MDA-MB-231 cells were grown in 15 cm dishes until 80% confluent. For glucose stimulation, cells were glucose-deprived for 3 hours by incubating in glucose-free DMEM supplemented with 10% FBS, followed by 4 hours stimulation with 5mM or 25mM glucose. Cells were crosslinked in 1% formaldehyde and quenched with 125mM glycine. Chromatin was sheared by sonication using a Branson Sonifier, precleared with control IgG antibodies and agarose beads (Millipore), and then immunoprecipitated with IgG (control), SRC-3, pSRC-3-S857 and ATF4 antibodies. DNA fragments were eluted from beads followed by reverse-crosslinking and purified DNA was used in qPCR reactions using SYBR green (Applied Biosytems) to determine the promoter occupancy. Melt curve analysis was performed to verify all SYBR green reactions produced a single PCR product.
Psrc 3 s857
Manufactured by Merck Group
The PSRC-3-S857 is a specialized laboratory equipment designed for scientific research and experimentation. It serves as a precision tool for carrying out various analytical and experimental procedures. The core function of this product is to perform specific tasks required in the laboratory setting, without further interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
2 protocols using psrc 3 s857
1
Chromatin Immunoprecipitation Assay Protocol
2
Chromatin Immunoprecipitation Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used for ChIP: SRC-3 (Cell Signaling or BD Biosciences), ATF4 (Santa Cruz C-20, and Cat# 11815 Cell Signaling), pSRC-3-S857 (Cell Signaling), and rabbit IgG. ChIP assays were performed according to an EZ ChIP kit (Millipore) with some modification35 (link). Briefly, MDA-MB-231 cells were grown in 15 cm dishes until 80% confluent. For glucose stimulation, cells were glucose-deprived for 3 hours by incubating in glucose-free DMEM supplemented with 10% FBS, followed by 4 hours stimulation with 5mM or 25mM glucose. Cells were crosslinked in 1% formaldehyde and quenched with 125mM glycine. Chromatin was sheared by sonication using a Branson Sonifier, precleared with control IgG antibodies and agarose beads (Millipore), and then immunoprecipitated with IgG (control), SRC-3, pSRC-3-S857 and ATF4 antibodies. DNA fragments were eluted from beads followed by reverse-crosslinking and purified DNA was used in qPCR reactions using SYBR green (Applied Biosytems) to determine the promoter occupancy. Melt curve analysis was performed to verify all SYBR green reactions produced a single PCR product.
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!