Description
Human/Mouse 4EBP1 (S65) Phosphorylation ELISA Kit (BA0233) (BA0233)
Protein phosphorylation plays a key role in regulating protein activity within cells, and a large number of protein kinases and phosphatases are involved in signal transduction pathways. 4E-binding protein 1 (4EBP1, 4E-BP1, EIF4EBP1, BP-1, PHAS-I) is a eukaryotic translation initiation factor that is phosphorylated in response to signals including UV irradiation and insulin signalling, resulting in activation of mRNA translation. The Human/Mouse 4EBP1 (S65) Phosphorylation ELISA Kit (SKU: BA0233) provides a rapid and sensitive assay for measuring the phosphorylated protein p4EBP1(S65) in cultured cells and normalises the level of phosphorylation to the total protein content in the same well. This simple, efficient cell-based 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. Cells grown in a 96-well plate are fixed and permeabilised, and phosphorylation is measured using a specific primary antibody followed by an HRP-conjugated secondary antibody with a fluorogenic substrate, alongside a fluorescent reagent that measures total protein.
| Product Name: | Human/Mouse 4EBP1 (S65) Phosphorylation ELISA Kit (BA0233) |
| SKU: | BA0233 |
| Detection Method: | Fluorimetric cell-based ELISA (λex/em = 530/585 nm for p4EBP1(S65) and 360/450 nm for total protein) |
| Detection Range: | Cell-based phosphorylation status assay (normalised to total cellular protein) |
| Sample Type: | Cells (cultured whole cells) |
| Species Reactivity: | Human (S65), mouse/rat (S64) |
| Assay Time: | 6.5 hours (hands-on time 2.5 hours) |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | This kit is shipped on ice. Upon delivery, store all reagents at -20°C. |
| Shelf Life: | About 6 months after receipt. |
| Shipping: | Gel Pack |
A fluorimetric cell-based assay for 4EBP1(S65) phosphorylation status. Cells grown in a 96-well plate are fixed and permeabilised; p4EBP1(S65) is detected using a specific primary antibody, an HRP-conjugated secondary antibody and a fluorogenic substrate (read at 530/585 nm), while a protein stain measures total protein (read at 360/450 nm) for normalisation.
- 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 necessary.
- Accurate and high-throughput. Protein phosphorylation is normalised to total cellular protein in the same well, greatly minimising well-to-well variations. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.
- For determination of 4EBP1(S65) phosphorylation status in whole cells and evaluation of pathway modulation by activators, inhibitors, siRNA etc. Species tested: human (S65), mouse/rat (S64).
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Prepare 1x Wash Buffer by diluting Stock Wash Buffer 40-fold with dH2O; reserve 6 mL 1x Wash Buffer for the Detection step. Assay samples in triplicate or more, and include a Protein Blank (no cells) and a Sample Blank (cells, no Ab1). |
| 2 | Culture and treat cells: seed 100 µL of 1-3×10⁴ adherent cells (or 4-10×10⁴ suspension cells) per well of a black 96-well culture plate; add 100 µL cell-free media to three wells as Protein Blank. Incubate overnight at 37°C, then treat cells as desired. |
| 3 | Fix cells with 4% (adherent) or 8% (suspension) formaldehyde solution for 20 min at room temperature (handle formaldehyde with caution in a chemical hood). Wash twice with 200 µL 1x Wash Buffer with gentle shaking (3 min each). |
| 4 | Add 100 µL Quench Buffer (2.2 mL 3% H2O2 + 8.8 mL 1x Wash Buffer) per well; incubate 20 min at room temperature. Wash three times with 200 µL 1x Wash Buffer. |
| 5 | Add 100 µL Blocking Buffer; incubate 1 hr at room temperature. |
| 6 | Add primary antibody: prepare 55 µL Ab1 Mixture per well (Ab1 : Blocking Buffer, 1:1000). Add 50 µL Blocking Buffer to Sample Blank wells and 50 µL Ab1 Mixture to Sample wells. Incubate 90 min at room temperature (or overnight at 2-8°C) with gentle shaking. Wash three times. |
| 7 | Add secondary antibody: prepare 55 µL Ab2 Mixture per well (Ab2 : Blocking Buffer, 1:1000). Add 50 µL to all wells; incubate 90 min at room temperature with gentle shaking. Wash four times. |
| 8 | Detection: prepare HRP Substrate (60 µL Dye Reagent + 6 mL 1x Wash Buffer + 6 µL 3% H2O2). Add 50 µL per well; incubate 30 min at room temperature in the dark. |
| 9 | Add 50 µL Protein Stain per well; incubate a further 5 min in the dark. Read at λex/em = 530/585 nm for p4EBP1(S65) and 360/450 nm for total protein. |
Calculate mean p4EBP1(S65) fluorescence at 530/585 nm for the Sample Blank (F_pTarget.Blank) and Sample wells (F_pTarget.Sample), and mean protein fluorescence at 360/450 nm for the Protein Blank (F_Protein.Blank) and Sample wells (F_Protein.Sample). ∆F_pTarget = F_pTarget.Sample – F_pTarget.Blank; ∆F_Protein = F_Protein.Sample – F_Protein.Blank. Normalised pTarget = (∆F_pTarget / ∆F_Protein) / (∆F_pTarget / ∆F_Protein)_0, where the subscript 0 is the control reference value (e.g. time zero or untreated wells).
| Component | Quantity | Storage |
| Stock Wash Buffer | 25 mL | -20°C |
| Blocking Buffer | 25 mL | -20°C |
| Protein Stain | 6 mL | -20°C |
| Dye Reagent | 120 µL | -20°C |
| Ab1 | 10 µL | -20°C |
| Ab2 (gM-HRP) | 10 µL | -20°C |
