Anti his alexa488

Manufactured by Qiagen

Sourced in United States

The Anti-HIS-Alexa488 is a fluorescently-labeled antibody that specifically binds to histidine-tagged (HIS-tagged) proteins. It is used to detect and visualize HIS-tagged proteins in various applications, such as Western blotting, immunoprecipitation, and flow cytometry.

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Lab products found in correlation

Dm5500b fluorescence microscope, by Leica (1 mentions)

3 protocols using anti his alexa488

Quantifying Cell-Specific CEA Binding

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To evaluate binding capacity of the ssScFv fragments on living cells, tumor cells with high (HT-29) and low CEA expression (COLO-320) were seeded on chamber slides at a density of 40,000 cells per chamber in 200 uL of the corresponding medium. After 72 h, cells were incubated with the fluorescein conjugated anti CEA polyclonal antibody and various concentrations of the specific (ssSM3E/fluorescein) or control (F73/fluorescein) tracers for 1 h at room temperature. After incubation, cells were washed 2 times with 0.5% BSA/PBS to discard excess non-bound tracers. Cells were incubated with secondary antibodies anti-his/Alexa488 (35310 Qiagen 1/100) for 30 minutes at RT. Slides were washed two times and mounted using Vectashield with DAPI for nucleus staining. Fluorescent signals were detected using a Leica DM5500 B fluorescence microscope.

Boonstra M.C., Tolner B., Schaafsma B.E., Boogerd L.S., Prevoo H.A., Bhavsar G., Kuppen P.J., Sier C.F., Bonsing B.A., Frangioni J.V., van de Velde C.J., Chester K.A, & Vahrmeijer A.L. (2015). Pre-clinical evaluation of a novel CEA-targeting near-infrared fluorescent tracer delineating colorectal and pancreatic tumors. International journal of cancer, 137(8), 1910-1920.

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Quantifying Estrogen Receptor Alpha Binding

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This method has been described previously (Koppen et al, 2009 (link)). Cell lysates of each clone were prepared and ERα was quantified by ELISA (Active Motif, USA) to enable equimolar input. An array with a set of immobilized peptides representing coregulator-derived NR-binding motifs is incubated with a reaction mixture of crude lysate, vehicle (2% DMSO) with or without 1 μM 17-β-estradiol (E2) and anti-HIS-Alexa488 (Qiagen, USA). Incubation was performed for 40 minutes at 20 °C, followed by removal of unbound receptor by washing and generation of a tiff image of each array using a PamStation96 (PamGene International). Image processing and quantification of ERα binding to each peptide on the array was performed by Bionavigator software (PamGene International). The list of protein peptides evaluated detailed in table S8.

Jeselsohn R., Bergholz J.S., Pun M., Cornwell M., Liu W., Nardone A., Xiao T., Li W., Qiu X., Buchwalter G., Feiglin A., Abell-Hart K., Fei T., Rao P., Long H., Kwiatkowski N., Zhang T., Gray N., Melchers D., Houtman R., Liu X.S., Cohen O., Wagle N., Winer E.P., Zhao J, & Brown M. (2018). Allele-specific chromatin recruitment and therapeutic vulnerabilities of ESR1 activating mutations. Cancer cell, 33(2), 173-186.e5.

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Quantitative ER Coregulator Binding Assay

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This method has been described previously (Koppen et al., 2009 (link)). Cell lysates of MCF7 cells expressing HA-tagged WT-ER, Y537S mutant ER and D538G mutant ER were quantified by ELISA (Active Motif, USA) to enable equimolar input. An array with a set of immobilized peptides representing coregulator-derived NR-binding motifs was incubated with a reaction mixture of crude lysate, vehicle (2% DMSO) with or without 1 μM 17β-estradiol (E2), increasing concentrations of BZA, 4-OHT or FULV (0.1, 1, 10, 100, 1000 nM) and anti-HIS-Alexa488 (Qiagen, USA). Incubation was performed for 40 min at 20°C, followed by removal of unbound receptor by washing and generation of a TIFF image of each array using a PamStation96 (PamGene International). Image processing and quantification of ERα binding to each peptide on the array was performed by Bionavigator software (PamGene International).

Fanning S.W., Jeselsohn R., Dharmarajan V., Mayne C.G., Karimi M., Buchwalter G., Houtman R., Toy W., Fowler C.E., Han R., Lainé M., Carlson K.E., Martin T.A., Nowak J., Nwachukwu J.C., Hosfield D.J., Chandarlapaty S., Tajkhorshid E., Nettles K.W., Griffin P.R., Shen Y., Katzenellenbogen J.A., Brown M, & Greene G.L. (2018). The SERM/SERD bazedoxifene disrupts ESR1 helix 12 to overcome acquired hormone resistance in breast cancer cells. eLife, 7, e37161.

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