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Tubastatin alexa647 tracer

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Tubastatin‐Alexa647‐Tracer is a fluorescent dye-labeled compound used for the detection and analysis of target proteins in biological samples. It is designed to specifically bind to and label target proteins, enabling their visualization and quantification through fluorescence-based techniques such as Western blotting, immunocytochemistry, or flow cytometry.

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Lab products found in correlation

Proxiplate, by PerkinElmer (2 mentions) Gst hdac10, by Thermo Fisher Scientific (2 mentions) Lanthascreen eu anti gst, by Thermo Fisher Scientific (2 mentions)

2 protocols using tubastatin alexa647 tracer

1

HDAC10 Inhibition Assay via TR-FRET

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The FRET‐assay was performed in white 384‐well ProxiPlates (PerkinElmer). Reagents were diluted in assay buffer (50 mM HEPES pH 8.0, 150 mM NaCl, 10 mM MgCl2, 1 mM EGTA, 0.01 % Brij‐35). Final assay volume (10 μL) contains 3 nM GST‐HDAC10 (Life Technologies), 30 nM Tubastatin‐Alexa647‐Tracer,[32] and 0.5 nM LanthaScreen Eu‐anti‐GST (Life Technologies). Compound stocks (10 mM DMSO) were diluted in assay buffer. An 11‐fold 1 : 3‐serial dilution of test compounds (1 μL) were presented in the plate and complemented by 9 μL of assay mix. After incubation (1 h, room temperature) TR‐FRET was measured with EnVision plate reader (Ex: 3 flashes of the TRF‐europiumlaser; Em: 620 and 665 nm (665/620 nm ratio). Inhibition was calculated by using negative control (2 % DMSO) and positive control (20 μM vorinostat). Dose‐response curves were fitted in ActivityBase (IDBS) using a four‐parameter logistic model and IC50‐values were calculated.[32]
Herp D., Ridinger J., Robaa D., Shinsky S.A., Schmidtkunz K., Yesiloglu T.Z., Bayer T., Steimbach R.R., Herbst‐Gervasoni C.J., Merz A., Romier C., Sehr P., Gunkel N., Miller A.K., Christianson D.W., Oehme I., Sippl W, & Jung M. (2022). First Fluorescent Acetylspermidine Deacetylation Assay for HDAC10 Identifies Selective Inhibitors with Cellular Target Engagement. Chembiochem, 23(14), e202200180.
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2

HDAC10 Enzyme Inhibition Assay

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The FRET-assay was performed in white 384-well ProxiPlates (PerkinElmer). Reagents were diluted in assay buffer (50 mM HEPES pH 8.0, 150 mM NaCl, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35). Final assay volume (10 μL) contains 3 nM GST-HDAC10 (Life Technologies), 30 nM Tubastatin-Alexa647-Tracer[32 (link)], and 0.5 nM LanthaScreen Eu-anti-GST (Life Technologies). Compound stocks (10 mM DMSO) were diluted in assay buffer. An 11-fold 1:3-serial dilution of test compounds (1 μL) were presented in the plate and complemented by 9 μL of assay mix. After incubation (1 h, room temperature) TR-FRET was measured with EnVision plate reader (Ex: 3 flashes of the TRF-europiumlaser; Em: 620 and 665 nm (665/620 nm ratio). Inhibition was calculated by using negative control (2 % DMSO) and positive control (20 μM vorinostat). Dose-response curves were fitted in ActivityBase (IDBS) using a four-parameter logistic model and IC50-values were calculated.[32 (link)]
Herp D., Ridinger J., Robaa D., Shinsky S.A., Schmidtkunz K., Yesiloglu T.Z., Bayer T., Steimbach R.R., Herbst‐Gervasoni C.J., Merz A., Romier C., Sehr P., Gunkel N., Miller A.K., Christianson D.W., Oehme I., Sippl W, & Jung M. (2022). First Fluorescent Acetylspermidine Deacetylation Assay for HDAC10 Identifies Selective Inhibitors with Cellular Target Engagement. Chembiochem, 23(14), e202200180.
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