HTRF Human Total Cyclin E1 Detection Kit HTRF®

The Total Cyclin E1 kit is designed to monitor the expression level of cellular Cyclin E1, an oncogene overexpressed in many human cancers.

All inclusive kit
Low sample consumption
No-wash
High sensitivity

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The Total Cyclin E1 kit is designed to monitor the expression level of cellular Cyclin E1, an oncogene overexpressed in many human cancers.

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Overview

E-type cyclins play an important role in the transition of quiescent cells into the cell cycle. Two E-type cyclins have been previously described, Cyclin E1 and Cyclin E2. Cyclin E1 is known to activate CDK2 inducing S-phase transition and DNA synthesis associated with mitosis. Overexpression of Cyclin E1 is directly implicated in many cancers.

This kit is compatible with the buffer from Total & phospho-CDK2 kits (≠64CDK2TPEG & ≠ 64CDK2Y15PEG), so the same lysate can be used for analyses of the level of Total Cyclin E1, Total CDK2 and CDK2 phospho Tyr15.

Benefits

SPECIFICITY
PRECISION

Total Cyclin E1 upregulation using Hydroxyurea and Aphidicolin

HeLa cells were cultured in a 96-well plate (100,000 cells/well) for 6h, and then treated overnight with increasing concentration of Hydroxyurea (inducer of single strand breaks) or Aphidicolin (cell cycle blocker). After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF Total Cyclin E1 detection antibodies were added. The HTRF signal was recorded after a 3h incubation.

As expected, the levels of Total Cyclin E1 increased in presence of hydroxyurea or aphidicolin.

Total (h) Cyclin E1 modulation using Hydroxyurea

Total (h) Cyclin E1 modulation using Aphidicolin

Total Cyclin inhibtion using Nocodazole

HeLa cells were plated in complete culture medium in a 96-well culture-treated plate at 100,000 cells/well, and incubated for 6 hours at 37 °C, 5% CO2. The cells were next treated with increasing concentrations of Nocodazole for 16 hours, and then lysed with 50 µL of supplemented lysis buffer #2 (1X) for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a low volume white microplate and 4 µL of the Total Cyclin E1 detection reagents were added. The HTRF signal was recorded after 3 hours of incubation.

Cell treatment with Nocodazole (cell cycle blocker) induced a dose-dependent decrease in the cellular content of Cyclin E1.

The Nocodazole treatment had no effect on cell proliferation or viability, as evidenced by the constant ATP levels measured with ATPliteTM assay.

Total Cyclin E1 inhibition using Nocodazole

Specificity of HTRF (h) Total Cylin E1 assay using siRNA experiments

HeLa cells were plated in 96-well plates (50,000 cells/well) and cultured for 24h. The cells were then transfected with siRNAs specific for Cyclin E1 and Cyclin E2, as well as with a negative control siRNA.

After a 48h incubation, the cells were lyzed and 16 µL of lysates were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF Total Cyclin E1 detection antibodies. The HTRF signal was recorded after 3 hours of incubation.

Cell transfection with Cyclin E1 siRNA led to an 80% signal decrease compared to the cells transfected with the negative siRNA. No signal decrease was observed for cells transfected with Cyclin E2 siRNA. The data demonstrate that the HTRF Total Cyclin E1 assay is specific for the detection of Cyclin E1 protein and does not cross-react with Cyclin E2.

Specificity of HTRF (h) Total Cylin E1 assay using siRNA experiments

HTRF (h) Total Cyclin E1 assay versatility on human cell lines

The adherent Human cell lines HeLa, MCF7, U20S, or HAP1 cells were seeded at 100,000 cells / well in a 96-well microplate. After a 24h incubation, the cells were lyzed with supplemented lysis buffer, and 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF Total Cyclin E1 detection reagents. The HTRF signal was recorded after 3 hours of incubation.

The HTRF Total Cyclin E1 assay efficiently detects Cyclin E1 in various cellular models expressing different levels of the protein.

HTRF (h) Total Cyclin E1 assay Versatility on human cell lines

HTRF Total Cyclin E1 assay compared to Western Blot

U2OS cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After a 48h incubation and cell medium removal, the cells were lysed with 3 mL of supplemented lysis buffer #2 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer#2, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF Total Cyclin E1 detection reagents.

Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

In these conditions, the HTRF Total Cyclin E1 assay was 16-fold more sensitive than the Western Blot technique.

Comparison between HTRF and WB sensitivity on Total Cyclin E1

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

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Insider Tips for successful sample treatment - Technical Notes

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HTRF and WB compatible guidelines - Technical Notes

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Protocol for tumor xenograft analysis with HTRF - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

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Multi-tissue cellular modeling and anlysis of insulin signaling - Posters

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A solution for phospho-protein analysis in metabolic disorders - Posters

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Detailed protocol and direct comparison with WB - Posters

Implement HTRF phospho-protein assays at every step of the drug discovery process, from in vitro to in vivo models

A single technology for 2D cells, 3D cells, and xenograft models - Posters

Simplify complex pathway dissection by combining the power of HTRF® cellular phospho-assays and the flexibility of the CyBi®-FeliX liquid handling system

PI3K/AKT/mTor translational control pathway - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery

One technology across all samples - Application Notes

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

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Properly interpret your compound effect - Application Notes

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening

In collaboration with Bayer - Scientific Presentations

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Product Insert Cyclin E1 Total Kit / 64CYCE1TPEG-64CYCE1TPEH

64CYCE1TPEG-64CYCE1TPEH - Product Insert

Safety Data Sheet (DEU) Cyclin E1 Total Kit / 64CYCE1TPEG