ChIP assay protocols were previously detailed [22 (link)]. In brief, cell lysates were sonicated using a Misonix Sonicator 3000 Homogenizer, resulting in fragmented genomic DNA. These lysates, diluted in ChIP dilution buffer, were subjected to immunoprecipitation with an anti-GATA4 antibody (Abcam). The DNA bound by GATA4 was eluted from protein A/G agarose, and NaCl was added to reverse cross-linking between proteins and genomic DNA. The DNA containing the proposed conserved Gαi3 promoter site [51 (link)] was subsequently analyzed via quantitative PCR (qPCR).
Anti gata4 antibody
Manufactured by Abcam
Anti-GATA4 antibody is a laboratory reagent used to detect and study the GATA4 protein, a transcription factor involved in the regulation of gene expression. This antibody can be used in various research applications, such as Western blotting, immunohistochemistry, and immunocytochemistry, to identify and quantify the presence of GATA4 in biological samples.
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3 protocols using anti gata4 antibody
1
ChIP Assay for GATA4 Binding
2
Antibody Reagents and Cell Culture
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Polybrene and puromycin were provided by Sigma-Aldrich (St. Louis, MO). The anti-GATA4 antibody was purchased from Abcam. The phosphorylated serine antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Other antibodies, as reported previously (20 (link), 31 (link)), were provided by Cell Signaling Technology (Beverly, MA) and SignalAntibody (Shanghai, China). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), antibiotics, and other cell culture reagents were provided by Gibco-BRL (Suzhou, China).
3
GATA-4 Expression in Isolated Germ Cells
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Isolated germ cells were fixed with methanol for 40 min and 1 % BSA to block unspecific sites was added, and then they were incubated for 45 min at RT with 0.1 % Triton X-100 and 10 % FBS for permeabilization. Later, cells were incubated with primary anti-GATA-4 antibody
(1:1200) (Rabbit monoclonal; Abcam Inc., Cambridge, MA) overnight at 4 ºC. The next day, the cells were washed and incubated with the secondary antibody (1:2000) (Goat Anti-Rabbit IgG-Alexa Fluor 488; Abcam Inc., Cambridge, MA) for 1 h at RT. Finally, the data from approximately 5,000 events were collected using an LSR Fortessa Flow Cytometer (Becton Dickinson, Mountain View, CA). HepG2 cells were used as a positive control for the assay under the same conditions.
(1:1200) (Rabbit monoclonal; Abcam Inc., Cambridge, MA) overnight at 4 ºC. The next day, the cells were washed and incubated with the secondary antibody (1:2000) (Goat Anti-Rabbit IgG-Alexa Fluor 488; Abcam Inc., Cambridge, MA) for 1 h at RT. Finally, the data from approximately 5,000 events were collected using an LSR Fortessa Flow Cytometer (Becton Dickinson, Mountain View, CA). HepG2 cells were used as a positive control for the assay under the same conditions.
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