EnzyFluo™ Human/Mouse 4EBP1(S65) Phosphorylation ELISA Kit

Protocol SDS

Application

• For determination of 4EBP1(S65) phosphorylation status in whole cells and evaluation of pathway modulation by activators, inhibitors, siRNA etc.

Key Features

• New and improved. Total assay time reduced from the standard 21 hours to 6.5 hours (hands-on time 2.5 hrs).
• Simple and convenient. Cells are directly cultured in 96-well plates. No cell lysis is necessary.
• Accurate and high-throughput. Protein phosphorylation is normalized to total cellular protein in the same well, greatly minimizing well-to-well variations. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.

Method

• Cell-Based ELISA (FL530/585 nm, 360/450 nm)

Samples

• Cells

Species

• human (S65), mouse/rat (S64)

Procedure

• The assay takes 6.5 hrs, hands-on time 2.5 hrs

Size

• 100 tests

Shelf Life

• 6 months

More Details

PROTEIN PHOSPHORYLATION plays a key role in regulating protein activity within cells. A large number of protein kinases and phosphatases are known to be involved in a variety of signal transduction pathways. A rapid assay capable of monitoring such signal transduction pathways, target protein phosphorylation status, and drug effects are highly desirable in life science research and drug discovery.4E-Binding Protein 1 (4EBP1, 4E-BP1, EIF4EBP1, BP-1, PHAS-I) is a eukaryotic translation initiation factor that is phosphorylated in response to various signals including UV irradiation and insulin signaling, resulting in activation of mRNA translation. BioAssay Systems’ 4EBP1(S65) Phosphorylation ELISA Kit provides a rapid and sensitive assay for measuring the phosphorylated protein p4EBP1(S65) in cultured cells and normalizes the level of phosphorylation to the total protein content in the same well. This simple and efficient assay eliminates the need for cell lysate preparation and can be used to study the underlying signal pathway and the effects of inhibitors, siRNA, or activators on the pathway. In this assay, cells grown in a 96-well plate are fixed and permeabilized. The phosphorylation of 4EBP1(S65) is measured using a specific primary antibody followed by a HRP-conjugated secondary antibody with a fluorogenic substrate, and a fluorescent reagent that measures the total protein in the same well.

Haven’t Found the ELISA Kit for Your Target

We offer two DIY kits: EFC1M-100 for mouse antibodies and EFC2R-100 for rabbit antibodies. All that is required is a primary antibody. If you prefer, we can develop the assay for you. Please email or call 1-510-782-9988 x 2 to request assay service.