Ar c 19

Manufactured by Santa Cruz Biotechnology

Sourced in United States, Canada

The AR C-19 is a laboratory instrument designed for the detection and analysis of specific antigens. It utilizes advanced immunoassay technology to provide accurate and reliable results. The core function of the AR C-19 is to facilitate the identification and quantification of target analytes in biological samples.

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Lab products found in correlation

Ar n 20, by Santa Cruz Biotechnology (3 mentions) Ing4 ep3804, by Abcam (2 mentions) Gapdh 6cs, by Merck Group (2 mentions) Confocal microscope, by Zeiss (1 mentions) Fugene 6, by Promega (1 mentions) Ar v7, by GeneTex (1 mentions) Gfp ab290, by Abcam (1 mentions) Ki 67 clone mm1, by Leica (1 mentions) Yap g 6, by Santa Cruz Biotechnology (1 mentions) Parp1 2 clone h250, by Santa Cruz Biotechnology (1 mentions) α tubulin clone dm1a, by Merck Group (1 mentions) Ar antibody, by Active Motif (1 mentions) Creb d76d11, by Cell Signaling Technology (1 mentions) Pten 138g6, by Cell Signaling Technology (1 mentions) Itgα6 goh3, by BD (1 mentions) Sybr green qpcr kit, by Thermo Fisher Scientific (1 mentions) 10 cm plates, by Thermo Fisher Scientific (1 mentions) Protein a sepharose, by GE Healthcare (1 mentions) Dual luciferase assay reagent, by Promega (1 mentions) Lipofectamine, by Thermo Fisher Scientific (1 mentions)

12 protocols using ar c 19

Immunofluorescence Analysis of Androgen Receptor

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COS1 cells were plated at 30% confluence on cover slips in 24 well plates. Cells were transfected with pSVAR and pGFP-MAD_7-35AR_1-54 using FuGENE 6 (Promega, WI, USA) and incubated for 24hrs before treatment with ligand for 2hrs. Wells were washed 3x with PBS and incubated in 1% paraformaldehyde for 10 min, washed 3x in PBS and incubated for a further 10 min in 0.1% Triton X-100 in PBS. Wells were again washed 3x with PBS, incubated in blocking solution (5% BSA in PBS) for 30 min and a further 1 hr with an AR antibody (AR C19, Santa Cruz Biotechnology, TX, USA) diluted 1:200 in blocking solution. Wells were washed 3x with PBS, re-blocked and incubated for 1hr with 594-Alexa Fluor conjugated secondary antibody (Invitrogen, Paisley, UK). A final 3x PBS washes was performed before the coverslips were mounted onto glass slides (Vectorshield containing DAPI, DAKO, Cambridge, UK). Images were obtained using a Zeiss Confocal Microscope, as previously described [46 (link)].

Brooke G.N., Powell S.M., Lavery D.N., Waxman J., Buluwela L., Ali S, & Bevan C.L. (2014). Engineered repressors are potent inhibitors of androgen receptor activity. Oncotarget, 5(4), 959-969.

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Antibody Characterization for ChIP and IP

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An AR antibody (RRID: AB_2793341) that recognises the N-terminus of AR was used by Active Motif for ChIP-exo analysis; this antibody also was used for ChIP-qPCR. Other antibodies used in the ChIP-qPCR assays include, AR-N20 (RRID:AB_1563391) Santa Cruz Biotechnology, Santa Cruz, CA, AR C-19 (RRID:AB_630864), rabbit anti mouse IgG (RRID:AB_228419). These antibodies and FOXA1 (RRID:AB_2104842), HOXB13 (GTX129245, GeneTex, Irvine, CA), Tubulin (RRID:AB_309885), Actin (RRID:AB_11004139), and AR-V7 (RRID:AB_2631057) antibodies were used for western blotting also. AR441 was used for western blotting and immunoprecipitation. It was grown in the BCM Protein and Monoclonal Antibody Production core, but also is sold as RRID AB_11000751 by ThermoFisher Scientific. A FOXA1 ChIP validated antibody (RRID:AB_2104842) was used for CoIP experiments.

Basil P., Robertson M.J., Bingman WE I.I.I., Dash A.K., Krause W.C., Shafi A.A., Piyarathna B., Coarfa C, & Weigel N.L. (2022). Cistrome and transcriptome analysis identifies unique androgen receptor (AR) and AR-V7 splice variant chromatin binding and transcriptional activities. Scientific Reports, 12, 5351.

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Androgen Receptor Chromatin Immunoprecipitation

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LNCaP-s17 cells were treated as indicated. DNA-AR protein complexes were cross-linked inside the cells by the addition of 1% formaldehyde. Whole-cell extracts were prepared by sonication, and an aliquot of the cross-linked DNA-protein complexes was immunoprecipitated by incubation with the AR-specific antibody (AR-C19; Santa Cruz Biotechnology) overnight at 4°C with rotation. Chromatin-antibody complexes were isolated from solution by incubation with protein A/G agarose beads for 1 hour at 4°C with rotation. The bound DNA-protein complexes were washed and eluted from beads with elution buffer (1% SDS and 0.1 mol/L NaHCO3), cross links were reversed, and DNA was extracted. The resulting chromatin preparations were analyzed by PCR using primers spanning either the proximal or the distal enhancer AREs of the PSA promoter. Primers used for ChIP assay were: ARE: 5’-CCTAGATGAAGTCTCCATGAGCTACA, ARE 3’-GGGAGGGAGAGCTAGCACTTG. Isotype-matched IgG was used as control (11 (link)).

Liu C., Lou W., Armstrong C., Zhu Y., Evans C.P, & Gao A.C. (2015). Niclosamide suppresses cell migration and invasion in enzalutamide resistant prostate cancer cells via Stat3-AR axis inhibition. The Prostate, 75(13), 1341-1353.

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Comprehensive Immunohistochemistry Protocol

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AR (C-19, sc-815, Santa Cruz Biotechnology and clone G122-434, BD), PSA (A0562, Dako), IKBKE (D20G4, Cell Signalling), α-tubulin (clone DM1A, T9026, Sigma), LATS2 (kpm C-2, sc-515579 Santa Cruz Biotechnology), YAP (G-6, sc-376830 Santa Cruz Biotechnology), c-MYC (ab56, Abcam and N262, sc-764, Santa Cruz Biotechnology), TMPRSS2 (H-4, sc-515727, Santa Cruz Biotechnology), PARP1/2 (clone H250, sc-7150, Santa Cruz Biotechnology), FKBP5 (D-4, sc-271547, Santa Cruz Biotechnology), GFP (ab290, AbCam) Ki67 (clone MM1, Novocastra, Leica Biotechnology).

Bainbridge A., Walker S., Smith J., Patterson K., Dutt A., Ng Y.M., Thomas H.D., Wilson L., McCullough B., Jones D., Maan A., Banks P., McCracken S.R., Gaughan L., Robson C.N, & Coffey K. (2020). IKBKE activity enhances AR levels in advanced prostate cancer via modulation of the Hippo pathway. Nucleic Acids Research, 48(10), 5366-5382.

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ChIP-qPCR Analysis of AR and AR-V7 Binding

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DNA-AR protein complexes were cross-linked inside the cells by the addition of 1% formaldehyde. Whole-cell extracts were prepared by sonication, and an aliquot of the cross-linked DNA-protein complexes was immunoprecipitated by incubation with antibodies against AR (AR-C19; Santa Cruz Biotechnology) or AR-V7 (AG10008, precision antibody) overnight at 4°C with rotation. Chromatin-antibody complexes were isolated from solution by incubation with protein A/G agarose beads for 1 hour at 4°C with rotation. The bound DNA-protein complexes were washed and eluted from beads with elution buffer (1% SDS and 0.1 mol/L NaHCO3), crosslinking was reversed, and DNA was extracted. The resulting chromatin preparations were analyzed by PCR using primers spanning either the proximal or the distal enhancer AREs of the PSA promoter (24 (link), 26 (link)). Isotype-matched IgG was used as control.

Liu C., Armstrong C.M., Lou W., Lombard A.P., Cucchiara V., Gu X., Yang J.C., Nadiminty N., Pan C.X., Evans C.P, & Gao A.C. (2017). Niclosamide and bicalutamide combination treatment overcomes enzalutamide and bicalutamide resistant prostate cancer. Molecular cancer therapeutics, 16(8), 1521-1530.

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ChIP-seq and ChIP-exo Protocols for Mapping AR and AR-V7 Binding

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ChIP assays were performed as previously described^{25 (link)} with the modifications using AR antibody (ActiveMotif, Carlsbad, CA) or AR C-19 (Santa Cruz Biotechnology, Dallas, TX) as described in the Supplementary methods (SMethods). Data generated by Active Motif using the ChIP-exo method and their AR antibody (ActiveMotif, Carlsbad, CA)^{53 (link)} were used to map AR and AR-V7 binding ChIP-seq and ChIP-exo (STable 1) were analysed as described in SMethods, using the bowtie2 (version 2.3, default parameters) alignment software^{54 (link)} and peaks were called using macs2 (version 2.1; − q < 0.05, otherwise mentioned)^{55 (link)}.

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Immunofluorescence and Immunoblotting Reagents

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Immunofluorescence: AR (C-19) and Miz1 (H-190) were purchased from Santa Cruz. ITGα6 (GoH3) antibody was purchased from BD Pharmingen. Immunoblotting: Myc (N-term) and ING4 (EP3804) antibodies were purchased from Abcam. Polyclonal Miz1 antibody was purchased from GeneTex. Tubulin antibody (DM1A) was purchased from Sigma and GAPDH (6CS) from Millipore.

Berger P.L., Winn M.E, & Miranti C.K. (2016). Miz1, a Novel Target of ING4, Can Drive Prostate Luminal Epithelial Cell Differentiation. The Prostate, 77(1), 49-59.

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Immunofluorescence and Western Blot Protocol

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Immunofluorescence and IHC: AR (C-19) and p63 (BC4A4) were purchased from Santa Cruz. ITGα6 (GoH3) was purchased from BD Pharmingen, ING4 polyclonal antibody was purchased from ProteinTech. K18 (CY-90) came from Sigma and K5/14 (HMW-34βE12/M0630) from DAKO. (pCREB/pATF1-Ser 133(87G3), CREB (D76D11), cleaved Caspase3 (Asp175)(5A1E), PSA (D11E1), and PTEN (138G6) were purchased from Cell Signaling Technology. Immunoblotting: ING4 (EP3804) and ATF1 (EPR1590) antibodies were purchased from Abcam. pCREB-Ser 133(87G3), CREB (D76D11) and PTEN (138G6) were purchased from Cell Signaling Technology. Tubulin antibody (DM1A) was purchased from Sigma and GAPDH (6CS) from Millipore. Chromatin Immunoprecipitations: ING4 (EP3804) was purchased from Abcam and CREB (D76D11) was purchased from Cell Signaling.

Watson M.J., Berger P.L., Banerjee K., Frank S.B., Tang L., Ganguly S.S., Hostetter G., Winn M, & Miranti C.K. (2021). Aberrant CREB1 Activation in Prostate Cancer Disrupts Normal Prostate Luminal Cell Differentiation. Oncogene, 40(18), 3260-3272.

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AR Transcriptional Regulation in LNCaP Cells

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LNCaP (1x10⁶ cells) stably transfected with vector or AR_1-558 were plated in 10cm plates (Nunc, Rochester, NY). The next day the media was replaced with 9 ml of serum-free RPMI. The cells were treated for 30 mins with R1881 (10nM), cross-linked with 1% formaldehyde and harvested. The cells were lysed in SDS lysis buffer (1%SDS, 50nM Tris (pH 8.0), 10mM EDTA, Complete™ protease inhibitors), and sonicated. The extracts were used for immunoprecipitation with anti-AR antibody AR (C-19; Santa Cruz Biotechnology). PSA primers used for real time PCR with Sybr green qPCR kit from (Invitrogen) were: 5’-GCCTGGATCTGAGAGATATCATC-3’ (forward) and 5’-ACACCTTTTTTTTCTGGA TTGTTG-3’ (reverse) [5 (link)].

Myung J.K., Wang G., Chiu H.H., Wang J., Mawji N.R, & Sadar M.D. (2017). Inhibition of androgen receptor by decoy molecules delays progression to castration-recurrent prostate cancer. PLoS ONE, 12(3), e0174134.

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Androgen Receptor Signaling Assay

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Androgens, [³H] mibolerone and R1881, were procured from Perkin Elmer (Waltham, MA), while lipofectamine, TaqMan PCR primers and fluorescent probes, master mixes, and Cells-to-Ct reagents were obtained from Life Technologies (Carlsbad, CA). Dual luciferase assay reagents were purchased from Promega (Madison, WI). Dihydrotestosterone (DHT), cell culture medium, and charcoal-stripped fetal bovine serum (csFBS) were purchased from Fisher Scientific (Waltham, MA). FBS was purchased from Hyclone (San Angelo, TX). AR-N20 and AR-C19 antibodies were procured from Santa Cruz biotechnology (Santa Cruz, CA). Enzalutamide was purchased from MedKoo Biosciences (Chapel Hill, NC). Protein A sepharose was procured from GE Healthcare (Pittsburg, PA). MG-132 was purchased from R&D and bortezomib was obtained from Selleckchem (Houston, TX). All other reagents used were analytical grade. Structure and purity of Enzalutamide were confirmed by NMR and mass spectrometry (Supplemental text).

Ponnusamy S., Coss C.C., Thiyagarajan T., Watts K., Hwang D.J., He Y., Selth L.A., McEwan I.J., Duke C.B., Pagadala J., Singh G., Wake R.W., Ledbetter C., Tilley W.D., Moldoveanu T., Dalton J.T., Miller D.D, & Narayanan R. (2017). Novel selective agents for the degradation of androgen receptor variants to treat castration-resistant prostate cancer. Cancer research, 77(22), 6282-6298.

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