Human c-Myc Cell-based Assay Kit

Human c-Myc assay kit

Based on the TR-FRET homogeneous robust HTRF® technology, the kit is designed for the rapid detection of human cMyc proto-oncongene in cells. This kit is the first solution to detect the endogenous cMyc using a cell-based assay. The assay enables high throughput without any washing steps, thereby saving time compared to ELISA. The simple protocol ensures quick assays, with results obtained after only 3 hours. This procedure is suitable for miniaturization.

Assay principle

HTRF® - THE HOMOGENEOUS CELL-BASED SANDWICH IMMUNOASSAY

Human c-Myc is measured sandwich immunoassays involving two specific anti-human c-Myc antibodies, respectively labelled with Europium cryptate (donor) and d2 (acceptor). The intensity of the signal is proportional to the concentration of c-Myc present in the sample.. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

After cell lysis, c-Myc can be quantitatively detected using the HTRF c-Myc assay kit reagents and most TR-FRET multimode plate readers.

A SIMPLER, MORE FLEXIBLE ASSAY PROTOCOL - ADAPTED TO YOUR APPLICATIONS

TWO-PLATE ASSAY PROTOCOL

The human c-Myc assay protocol is described here. Cells are plated, stimulated and next lysed in the same 96-well culture plate. Lysates are then transferred to the assay plate for the detection of human c-Myc by HTRF® reagents. This protocol enables the cells' viability and confluence to be monitored. The antibodies labelled with HTRF fluorophores may be pre-mixed and added in a single dispensing step, to further streamline the assay procedure. The assay detection can be run in 96- to 384-well plates by simply resizing each addition volume proportionally.

Product performance

1- C-MYC DETECTION IN SEVERAL HUMAN CELL LINES

100,000 human MCF7, A549, HeLa, HTC116 and HEK293T cells were plated in 96 well plates in cell culture medium and incubated for 24h at 37 °C - 5% CO2. Cell culture medium was then removed and cells were lysed with 50 µL of lysis buffer for 30 min at RT under gentle shaking. 10 µL of lysate was transferred into a 384-well sv white microplate, and 10 µL of the HTRF c-Myc detection reagents was added. The HTRF signal was recorded after an overnight incubation time.

2- INHIBITORY EFFECT OF JQ1, A SPECIFIC INHIBITOR OF BROMODOMAIN INTERACTION*

40,000 HeLa cells were plated in 96 well plate in cell culture medium, and incubated for 24h, at 37°C-5%CO2. After incubating with increasing concentrations of JQ1 for 24 hours, the medium was removed and cells were lysed with 50 µL of lysis buffer for 45 min at RT under gentle shaking. 10 µL of lysate was transferred into a 384-well sv white microplate and 10 µL of the HTRF c-Myc detection reagents was added. HTRF signal was recorded after an overnight incubation

* Ref: Delmore et al. Cell 2011

3- INHIBITORY EFFECT OF TSA, A SPECIFIC INHIBITOR OF HISTONE DEACETYLASE (HDAC)*

40,000 HeLa cells were plated in 96 well plate in cell culture medium, and incubated for 24h, at 37°C-5%CO2. After incubating with increasing concentrations of TSA for 24 hours, the medium was removed and cells were lysed with 50 µL of lysis buffer for 45 min at RT under gentle shaking. 10 µL of lysate was transferred into a 384-well sv white microplate and 10 µL of the HTRF c-Myc detection reagents was added. HTRF signal was recorded after an overnight incubation

* Ref: Li at Wu, Biochem Biophys Res Comm, 2004

4- INHIBITORY EFFECT OF U0126, METHOTREXATE AND GEMCITABINE, INHIBITORS OF CELL GROWTH

10,000 HeLa or A549 cells were plated in 96 well plate in cell culture medium, and incubated for 24h, at 37°C-5%CO2. After incubating with increasing concentrations of inhibitors (U0126, Methotrexate and Gemcitabine) for 72 hours, the medium was removed and cells were lysed with 50 µL of lysis buffer for 45 min at RT under gentle shaking. 10 µL of lysate was transferred into a 384-well sv white microplate and 10 µL of the HTRF c-Myc detection reagents was added. HTRF signal was recorded after an overnight incubation.

5- HTRF ASSAY COMPARED TO WESTERN BLOT

HeLa cells were grown in a T175 flask at 37 °C, 5% CO2 for 48 h. After medium removal, the cells were lysed with 3 mL of supplemented lysis buffer for 45 min at room temperature. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 10 µL of each dilution were dispensed and analyzed side-by-side by Western-blot and by HTRF.

By using HTRF c-Myc only 1,000 cells are sufficient for minimal signal detection while 4,000 cells are needed for a Western Blot signal. The HTRF c-Myc assay is at least 4-fold more sensitive than the Western Blot.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Human c-Myc cell-based kit - 500 tests63ADK053PEG
-
Human c-Myc cell-based kit - 10,000 tests63ADK053PEH

Companion products

DescriptionCat. noProduct insertMSDS
Human c-Myc cell-based kit control lysate63ADK053TDA
-
-
Lysis buffer 4 - 16 ml64LB4FDD
-
Lysis buffer 4 - 130 ml64LB4FDF
-

ドキュメント