Aryl hydrocarbon Receptor antibody [RPT9] (ab2769)

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Anonymous Abreview submitted on 3 September 2008
Application:	Immunoprecipitation
Aryl hydrocarbon Receptor antibody [RPT9] (ab2769)
Sample	Human Cell lysate - whole cell (Breast cancer, cell type)
Total protein in input:	50 µg
Specification	Breast cancer, cell type
Treatment	10ng/mL depsipeptide for 24hrs
Immuno- precipitation step	Protein A/G
Primary Antibody
Dilution	1/100
Incubation time:	16 hours  Temperature: 4°C Diluent: RIPA buffer
Western Blot Antibody
Name:	Non-Abcam Antibody was used: Rabbit anti-HSP90 polyclonal antibody (from Stress Conjugation: HRP
Dilution:	1/1000
Additional Data
Rating	Good
Additional notes	Upper panel (probed with anti-HSP90 antibody from Stressgen SPA-846)): This is a trial experiment to evaluate the association of AhR with HSP90 in MCF7 human breast cancer cell line (with or within treatment with 10ng/mL depsipeptide). Two mouse monoclonal anti-AhR antibodies were tried: [RPT9 (ab2769)] and [RPT1 (ab2770)] but RPT1 did not give any pull down of HSP90 in the cell lysate. Lane 1: protein marker (precision plus protein standards from BIO-RAD) Lane 2-3: IP with RPT1 (2=untreated MCF7 3= depsipeptide treated MCF7) Lane 4-5: IP with RPT9 (4=untreated MCF7 5= depsipeptide treated MCF7) Lane 6-7: no antibody IP control Lane 8-9: Input (whole cell lysate from untreated MCF7 and depsipeptide treated cells without IP) HSP90 pulled down by RPT9 (90kDa) is indicated by the arrow Lower panel (probed with anti-AhR antibody (RPT1 ab2770)): This blot is probed with an anti-AhR antibody RPT1 (ab2770: good for WB) to demonstrate the amount of AhR protein pulled down by using RPT9 (ab2769) in the IP reaction. Lane 1: protein marker (precision plus protein standards from BIO-RAD) Lane 2-3: IP with RPT9 (2=untreated MCF7 3= depsipeptide treated MCF7) Lane 4-5: no antibody IP control Lane 6-7: Input (whole cell lysate from untreated MCF7 and depsipeptide treated cells without IP) AhR protein pulled down by RPT9 (observed 100kDa) is indicated by the arrow
Image	Aryl hydrocarbon Receptor antibody [RPT9] for IP (Human) Copyright image used under license

Anonymous Abreview submitted on 22 August 2008
Application:	ChIP
Aryl hydrocarbon Receptor antibody [RPT9] (ab2769)
Sample	Human Cell lysate - whole cell (colon cancer, cell type)
Specification	colon cancer, cell type
Type:	Cross-linking (X-ChIP) - 10 Mins 0 Secs
Detection step:	Semiquantitative PCR
Positive Control:	my samples
Negative Control:	No template control for the PCR
Primary Antibody
Dilution	1/100
Incubation time:	16 hours
Additional Data
Rating	Good
Additional notes	This is a trial experiment to evaluate the association of AhR to the ABCG2 promoter using the mouse monoclonal [RPT9] for the IP (ChIP assay). Semiquantitative PCR was performed to evaluate the relative association of AhR with the proximal ABCG2 promoter in a S1 colon cancer cell line without treatment, or treated with depsipeptide (10 ng/mL 24h) or benzo(a)pyrene (10uM 24h). Lane 1: DNA ladder (100 bp from promega) Inputs (lanes 2-4): S1 no treatment - serial dilution Inputs (lanes 5-7): S1 treated with depsipeptide - serial dilution Inputs (lanes 8-10): S1 treated with benzo(a)pyrene - serial dilution AhR ChIP (lane 11-13): use 1:1 diluted immunoprecipitate for PCR lane 11=S1 no treatment lane 12=S1 depsipeptide 10ng/mL lane 13= S1 benzo(a)pyrene 10uM AhR ChIP (lane 14-16): use 1:2 diluted immunoprecipitate for PCR same order as lanes 11-13 Lane 17: H2O control for PCR
Image	Aryl hydrocarbon Receptor antibody [RPT9] for ChIP (Human) Copyright image used under license