LNCaP or CWR22rv1 cells were transfected with or without siRNAs of AR, FOXM1 or non-targeting control, and the cells were harvested and used for ChIP assays. ChIP assays were performed using ChIP-IT Expression Chromatin Immunoprecipitation Kits (Active motif, Carlsbad, CA, USA). In brief, the cells were fixed with formaldehyde for 10 minutes, and the fixation reaction was quenched with Glycine Stop-Fix solution. The cells were lysed and sonicated until the desired lengths were achieved (100–500 bp). For the immunoprecipitation of formaldehyde cross-linked chromatin-protein complexes, the antibodies against FOXM1 were used or IgG was used as the negative control. The same amount of chromatin without antibody incubation was used as the input control. The samples were incubated on an end-to-end rotor for 3 hours at 4°C. The reaction mixtures were eluted following the manufacturer’s protocol. DNA was analyzed via semi-quantitative PCR. Primers spanning the FOXM1 binding sequence were designed, and primers spanning the regions lacking the FOXM1 binding sites were used as controls (for primer information see supplementary text). All ChIP experiments were repeated at least three times.
Chip it expression chromatin immunoprecipitation kits
Manufactured by Active Motif
Sourced in United States
The ChIP-IT Expression Chromatin Immunoprecipitation Kits are designed for the isolation and analysis of protein-DNA complexes from cells. The kits provide reagents and protocols for chromatin preparation, immunoprecipitation, and DNA purification.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using chip it expression chromatin immunoprecipitation kits
1
ChIP Assay of FOXM1 Binding
2
ChIP Assay Using TCF12 Antibody
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIP assay was performed using ChIP-IT Expression Chromatin Immunoprecipitation Kits (Active motif, Carlsbad, CA, USA). In short, formaldehyde was used to treat cells for 10 min, and Glycine Stop-Fix solution was used to quench the reaction. Next, we achieved 100–500 bp DNA fragments after lysing and sonicating the cells. The immunocomplexes were precipitated by using antibodies against TCF12 and normal serum IgG overnight and rotationally incubation at 4 °C. The 20% of chromatin without antibody incubation was used as the input control. Finally, the reaction mixtures were eluted by using beads according to the manufacturer’s instruction.
3
Chromatin Immunoprecipitation for Transcription Factor Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromatin Immunoprecipitation (ChIP) assay was performed by using ChIP-IT Expression Chromatin Immunoprecipitation Kits (Active motif, Carlsbad, CA, USA). Cells were treated by formaldehyde for 10 min, lysed by Glycine Stop-Fix solution, and sonicated to achieve 100∼500 bp DNA fragments. The immuno-complexes were precipitated using antibodies against RELA and normal serum IgG (as negative control) under overnight rotationally incubation at 4°C. 30% amount of chromatin without antibody incubation was used as the input control. The reaction mixtures were eluted using beads following the manufacturer’s protocol and then applied in the following quantitative PCR analysis.
4
Transcriptional Regulation of S100A8/A9 by PU.1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The co-expression of PU.1 and S100A8/S100A9 was evaluated by the online bioinformatics tool ChIPBase version 2.0 (http://rna.sysu.edu.cn/chipbase/ ). PU.1 binding motif was also searched on the promoter regions of S100A8/S100A9 using ChIPBase 2.0.
ChIP assay was performed to validate the binding of PU.1 with the promoter sequences of S100A8 or S100A9 using ChIP-IT Expression Chromatin Immunoprecipitation Kits (Active motif, Carlsbad, CA, USA). In brief, cells were fixed with 4% formaldehyde for 10 min. 100 ~500 bp DNA fragments were obtained from lysed cells by ultrasonic crushing. The immuno-complexes were precipitated using PU.1 antibody and normal serum IgG through rotational incubation at 4° C overnight. The same amount of chromatin not incubated with antibodies was used as the input control. Enrichment of promoter sequences of S100A8 or S100A9 was then detected with qPCR in the precipitated DNA.
Luciferase reporter gene assay was used to evaluate the transcription regulatory activity of PU.1 on S100A8/S100A9 genes. Vectors containing different truncated promoter sequences were cloned and inserted into pGL3-basic vector (Promega, Madison, WI, USA) with PU.1 at the upstream. The assay was performed according to the manufacturer’s instructions.
ChIP assay was performed to validate the binding of PU.1 with the promoter sequences of S100A8 or S100A9 using ChIP-IT Expression Chromatin Immunoprecipitation Kits (Active motif, Carlsbad, CA, USA). In brief, cells were fixed with 4% formaldehyde for 10 min. 100 ~500 bp DNA fragments were obtained from lysed cells by ultrasonic crushing. The immuno-complexes were precipitated using PU.1 antibody and normal serum IgG through rotational incubation at 4° C overnight. The same amount of chromatin not incubated with antibodies was used as the input control. Enrichment of promoter sequences of S100A8 or S100A9 was then detected with qPCR in the precipitated DNA.
Luciferase reporter gene assay was used to evaluate the transcription regulatory activity of PU.1 on S100A8/S100A9 genes. Vectors containing different truncated promoter sequences were cloned and inserted into pGL3-basic vector (Promega, Madison, WI, USA) with PU.1 at the upstream. The assay was performed according to the manufacturer’s instructions.
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!