Epr3864

Manufactured by Abcam

Sourced in United States

EPR3864 is a recombinant monoclonal antibody recognizing β-Actin. The antibody is generated using the VelocImmune® technology.

Automatically generated - may contain errors

Lab products found in correlation

Dako autostainer, by Agilent Technologies (2 mentions) Immobilon p, by Merck Group (2 mentions) Halt protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions) Bioruptor equipment, by Diagenode (2 mentions) Mp 7451, by Vector Laboratories (1 mentions) Sm803, by Agilent Technologies (1 mentions) S236784 2, by Agilent Technologies (1 mentions) Diaminobenzidine, by Agilent Technologies (1 mentions) Magenta, by Agilent Technologies (1 mentions) Rm 2111 rq, by Thermo Fisher Scientific (1 mentions) Matlab, by MathWorks (1 mentions) Imagescope, by Leica Biosystems (1 mentions) Nanozoomer 2.0 ht, by Hamamatsu Photonics (1 mentions) A2066, by Merck Group (1 mentions) Primer3plus, by Merck Group (1 mentions) Envision, by Agilent Technologies (1 mentions) Real target retrieval solution, by Agilent Technologies (1 mentions) 4 6 diamidino 2 phenylindole dapi nuclear stain, by Thermo Fisher Scientific (1 mentions) Alexa 555, by Thermo Fisher Scientific (1 mentions) α sma, by Merck Group (1 mentions)

16 protocols using epr3864

Quantifying ERG Expression in Tumor Samples

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Tumors from different treatment conditions (±HS, HSP70^−/−) were harvested 48 hours after treatment with 5-FU or PBS as a control. These tumors were placed in tissue storage (Miltenyi, 130-100-008) and then left for a minimum of 48 hours in a formalin bath to fix the tissues. After this step, tumors were included in paraffin and were then cut into 4 µm thick slices. The paraffin was then removed using the PT-link device (Agilent S236784‐2). Slides were labeled for 1 hour with the anti-ERG monoclonal antibody (1/200; Abcam, EPR3864), then stained with ImmPRESS HRP Goat Anti-Rabbit IgG (Peroxidase) for 30 min (Vector, MP-7451) to amplify the signal. Finally, slides were stained with DAB (Agilent, SM803 and DM827) and counterstained with hematoxylin (Enzo, ENZ‐ACC106) and were mounted and digitally scanned using the Nanozoomer HT2.0 device (Hamamatsu). Staining quantification was carried out using QuPath software (v.01.3 ref). The appropriate threshold to discriminate positive cells from negative was determined on positive controls (endothelial cells of large vessels). To analyze the whole cohort, we generated two scripts. The first one was dedicated to upload 12 rectangles of 160,000 µm². These areas were then randomly placed on slides by the investigator. The second script detected ERG-positive cells in those areas. Then the percentage of positive cells was calculated.

Pilot T., Fratti A., Thinselin C., Perrichet A., Demontoux L., Limagne E., Derangère V., Ilie A., Ndiaye M., Jacquin E., Garrido C., Ghiringhelli F., Chalmin F, & Rébé C. (2020). Heat shock and HSP70 regulate 5-FU-mediated caspase-1 activation in myeloid-derived suppressor cells and tumor growth in mice. Journal for Immunotherapy of Cancer, 8(1), e000478.

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Quantifying EGFR and BBB Integrity in HGG

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EGFR (RM-2111-RQ [Thermo Fisher Scientific]; secondary, SM805[Agilent Technologies]) immunohistochemistry and hematoxylin counterstaining were performed after heat-mediated antigen retrieval with Dako Autostainer (Agilent) along positive and negative controls. Double immunohistochemical staining of Claudin-5 (1:500, 34–1,600; Thermo Fisher) and ETS-related gene (1:1,000, EPR3864; Abcam) was performed on HGG tissue to assess blood–brain barrier (BBB) integrity (2 (link)). Immunoreactivity was visualized with diaminobenzidine (for EGFR and Claudin-5) and magenta (for ETS-related gene) chromogens (Dako) and scanned in NanoZoomer 2.0-HT (Hamamatsu Photonics). The percentage of pixels with moderate to strong staining was quantified with ImageScope (Aperio Technologies) as previously described (11 (link)). EGFR-positive tumor cells within tumor outlines were counted with a MATLAB (MathWorks) algorithm.

Zhou Q., van den Berg N.S., Kang W., Pei J., Nishio N., van Keulen S., Engelen M.A., Lee Y.J., Hom M., Vega Leonel J.C., Hart Z., Vogel H., Cayrol R., Martin B.A., Roesner M., Shields G., Lui N., Gephart M.H., Raymundo R.C., Yi G., Granucci M., Grant G.A., Li G, & Rosenthal E.L. (2022). Factors for Differential Outcome Across Cancers in Clinical Molecule-Targeted Fluorescence Imaging. Journal of Nuclear Medicine, 63(11), 1693-1700.

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Transient Transfection of 293T Cells

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Human 293T cells were seeded onto poly-L-lysine-coated 6-cm dishes (15 (link)) and transiently transfected by the calcium phosphate coprecipitation method (16 (link)) with 4.5 µg pBluescript KS⁺ and either 4.5 µg empty vector pEV3S or ERG-Myc-Flag expression plasmid. Thirty-six hours after transfection, cells were washed once with phosphate-buffered saline and cells were detached by a 5-min incubation in 40 mM HEPES (pH 7.4), 150 mM NaCl, 10 mM EDTA, after which cells were sprayed off by pipetting. Then, cells were collected by centrifugation and resuspended in 150 µl of 10 mM Tris, 30 mM Na₄P₂O₇ (pH 7.1), 175 mM NaCl, 50 mM NaF, 2 mM dithiothreitol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 2 µg/ml aprotinin, 1 µg/ml pepstatin, lysed for 30 min on ice and debris was removed by centrifugation (17 (link)). Extracts were frozen in liquid nitrogen and then stored at −80°C before use in DNA-binding assays. The presence of ERG in these extracts was assessed by western blotting (18 (link)) utilizing rabbit monoclonal ERG antibody (EPR3864; ab92513; Abcam), while total actin was detected with a rabbit polyclonal antibody (A2066; Sigma).

KIM T.D., SHIN S, & JANKNECHT R. (2016). ETS transcription factor ERG cooperates with histone demethylase KDM4A. Oncology Reports, 35(6), 3679-3688.

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Genomic DNA Amplification and Variant Analysis

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Mutations were visualized using IGV 2.0.(26 (link)) Genomic DNA from each focus underwent amplification using GenomePlex WGA2 (Sigma, St. Louis, MO, USA) and primers targeting a subset of variants were designed using Primer3Plus and obtained from IDT (Coralville, IA, USA). The PCR products were sequenced in the University of Chicago DNA Sequencing and Genotyping Facility. ERG IHC was performed at the HTRC using anti-ERG antibody EPR3864 (Abcam, Cambridge, MA, USA).

VanderWeele D.J., Brown C.D., Taxy J.B., Gillard M., Hatcher D.M., Tom W.R., Stadler W.M, & White K.P. (2014). Low-grade prostate cancer diverges early from high grade and metastatic disease. Cancer Science, 105(8), 1079-1085.

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Immunohistochemical Analysis of Brain Tissue

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Formalin-fixed paraffin-embedded brain sections (4 μm thick) were incubated with primary antibodies after heat mediated antigen retrieval. All immunohistochemical procedures were performed following preprogramed standard protocol on an automatized histostainer (Dako Autostainer, Agilent) with positive and negative controls in each operation. Immunoreactivity was visualization with diaminobenzidine and magenta chromogens (Dako EnVision). A staining index was calculated as the product of intensity and fraction of positive tumor cells using Aperio ImageScope (Leica)⁵¹. The immunoreactivity of human brain tissues were scored by two board-certified pathologists as previously described^{52 (link)}. Primary antibodies were: EGFR (prediluted, Thermo Scientific, RM-2111-RQ), Ki-67 (prediluted, Dako, GA62661-2), Claudin-5 (1:500, Thermo Scientific, 34–1600) and ERG (1:1000, Abcam, EPR3864). Secondary antibody: EnVision FLEX + rabbit (linker) (prediluted, Dako, SM805) for EGFR; no secondary antibody was used for all other antibodies.

Zhou Q., Vega Leonel J.C., Santoso M.R., Wilson C., van den Berg N.S., Chan C.T., Aryal M., Vogel H., Cayrol R., Mandella M.J., Schonig F., Lu G., Gambhir S.S., Moseley M.E., Rosenthal E.L, & Grant G.A. (2021). Molecular imaging of a fluorescent antibody against epidermal growth factor receptor detects high-grade glioma. Scientific Reports, 11, 5710.

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Immunofluorescence Detection of ERG and α-SMA

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FFPE sections (3 μm) were deparaffinized and heated to expose the hidden antigens using Real Target Retrieval Solution (Dako). Samples were stained for immunofluorescence using primary antibodies to detect ERG (monoclonal rabbit, clone EPR3864, 1:800, Abcam, Cambridge, UK) and α-SMA (monoclonal mouse, clone 1A4, 1:3000, Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies Alexa 647 and Alexa 555 were incubated (Invitrogen, Carlsbad, CA, USA) at room temperature. Finally, the slides were mounted with 4,6-diamidino-2-phenylindole (DAPI) nuclear stain (Invitrogen, Carlsbad, CA, USA). Negative controls, in which primary antibodies were omitted, did not give rise to any detectable labeling. Images were captured with a LSM710 Zeiss Confocal Microscope (Zeiss, Oberkochen, Germany) and almost five arbitrary fields (magnification 630×) for each sample were quantified using the analysis software Image-J (National Institute of Health, Bethesda, MD, USA).

Díaz R., Sandoval P., Rodrigues-Diez R.R., del Peso G., Jiménez-Heffernan J.A., Ramos-Ruíz R., Llorens C., Laham G., Alvarez-Quiroga M., López-Cabrera M., Ruiz-Ortega M., Bajo M.A, & Selgas R. (2020). Increased miR-7641 Levels in Peritoneal Hyalinizing Vasculopathy in Long-Term Peritoneal Dialysis Patients. International Journal of Molecular Sciences, 21(16), 5824.

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Protein Detection and Chromatin Interactions

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Rabbit anti-ERG monoclonal antibody (EPR3864, Abcam Inc. Cambridge, MA) and mouse anti-GAPDH monoclonal antibody (sc-32233, Santa Cruz Biotechnology) were used for western blot analysis. Rabbit anti-ERG polyclonal antibody (sc-354, Santa Cruz Biotechnology) was used for Chromatin Immunoprecipitation (ChIP) assay. Western blot analysis was using standard procedures.

Zhang Z., Lanz R.B., Xiao L., Wang L., Hartig S.M., Ittmann M.M., Feng Q, & He B. (2016). The tumor suppressive miR-200b subfamily is an ERG target gene in human prostate tumors. Oncotarget, 7(25), 37993-38003.

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Immunodetection of ERG and DUX4 Proteins

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Cytospins of pre-B cells were fixed with 4% paraformaldehyde and washed with phosphate-buffered saline (PBS), followed by a 30 minute incubation in a blocking/permeabilization solution of 10% normal goat serum (NGS)/0.1% Triton-X 100/PBS, and then incubated for one hour with primary ERG antibody (Abcam EPR3864) diluted in 3% NGS/0.1% Triton-X 100/PBS. Slides were washed three times in PBS, and then incubated for 45 minutes in 3% NGS/0.1% Triton-X 100/PBS containing a secondary antibody conjugated to Ig-Alexa Fluor 555 (Invitrogen). Slides were washed three times in PBS and mounted with Vectashield (Vector Labs) containing 4’-6-diamidino-2-phenylindol (DAPI). All steps were carried out at room temperature. Images were captured using a Nikon C2 confocal fluorescence microscope.
Immunoblotting of cell line and leukemic cell whole cell lysates was performed as previously described^{32 (link),53} using ERG N-terminus specific antibodies (C20 and H95, Santa Cruz Biotechnologies, Santa Cruz, CA), and a C-terminus specific rabbit monoclonal antibody (Abcam 133264). DUX4 immunoblotting was performed with a C-terminus specific (E5-5, ab124699, Abcam) and N-terminus specific (aa82–131, LS-C205474, Lifespan Biosciences, Inc.) antibodies.

Zhang J., McCastlain K., Yoshihara H., Xu B., Chang Y., Churchman M.L., Wu G., Li Y., Wei L., Iacobucci I., Liu Y., Qu C., Wen J., Edmonson M., Payne-Turner D., Kaufmann K.B., Takayanagi S.I., Wienholds E., Waanders E., Ntziachristos P., Bakogianni S., Wang J., Aifantis I., Roberts K.G., Ma J., Song G., Easton J., Mulder H.L., Chen X., Newman S., Ma X., Rusch M., Gupta P., Boggs K., Vadodaria B., Dalton J., Liu Y., Valentine M.L., Ding L., Lu C., Fulton R.S., Fulton L., Tabib Y., Ochoa K., Devidas M., Pei D., Cheng C., Yang J., Evans W.E., Relling M.V., Pui C.H., Jeha S., Harvey R.C., Chen I.M., Willman C.L., Marcucci G., Bloomfield C.D., Kohlschmidt J., Mrózek K., Paietta E., Tallman M.S., Stock W., Foster M.C., Racevskis J., Rowe J.M., Luger S., Kornblau S.M., Shurtleff S.A., Raimondi S.C., Mardis E.R., Wilson R.K., Dick J.E., Hunger S.P., Loh M.L., Downing J.R, & Mullighan C.G. (2016). DEREGULATION OF DUX4 AND ERG IN ACUTE LYMPHOBLASTIC LEUKEMIA. Nature genetics, 48(12), 1481-1489.

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Protein Analysis after Cell Knockdown

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After knockdown experiments, cells were lysed in Triton-X lysis buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0,5% Triton x-100, 1 mM PMSF, 1 mM DTT and 1× Halt protease inhibitor cocktail (Thermo Fisher Scientific), after which the lysates were sonicated four times for 30 s at medium power with Bioruptor equipment (Diagenode), and cellular debris was removed by centrifugation. Proteins were separated by polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Immobilon-P; Millipore).
Primary antibodies against AR (AR-441; NeoMarkers; dilution 1:200), ERG (EPR3864; Abcam; dilution 1:5000), and pan-actin (ACTN05; NeoMarkers; 1:10 000) were used and detected by anti-mouse HRP-conjugated antibody produced in rabbit (dilution 1:2000-1:5000; DAKO) or by anti-rabbit HRP-conjugated antibody produced in swine (dilution 1:5000; DAKO) and Clarity Western ECL Substrate (Bio-Rad) with autoradiography.

Kohvakka A., Sattari M., Shcherban A., Annala M., Urbanucci A., Kesseli J., Tammela T.L.J., Kivinummi K., Latonen L., Nykter M, & Visakorpi T. (2020). AR and ERG drive the expression of prostate cancer specific long noncoding RNAs. Oncogene, 39(30).

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Immunohistochemical Analysis of ERG and MYC

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IHC was performed on TMA sections as described previously (6 (link),18 (link)), using primary antibodies against ERG (Ventana Medical Systems, EPR3864, predilute; Tucson, AZ, USA) and MYC (Epitomics, clone Y69, 1:200 dilution; Burlingame, CA, USA). For each evaluable TMA core, IHC was scored semi-quantitatively by two study pathologists (A.M.U. and R.M.), based on nuclear staining intensity (0–3; i.e., negative, weak, moderate, or strong) and percentage of positive tumor cells (0–100), and an IHC product score was calculated (range = 0–300). For a given patient and tumor nodule, IHC product scores were averaged across evaluable TMA cores.

Udager A.M., De Marzo A.M., Shi Y., Hicks J.L., Cao X., Siddiqui J., Jiang H., Chinnaiyan A.M, & Mehra R. (2016). Concurrent nuclear ERG and MYC protein overexpression defines a subset of locally advanced prostate cancer: potential opportunities for synergistic targeted therapeutics. The Prostate, 76(9), 845-853.

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