Eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) (eIF4E-binding protein 1) (Phosphorylated heat- and acid-stable protein regulated by insulin 1) (PHAS-I)
Quantitative/Semi-Quantitative
Semi-Quantitative
Specificity
The antibody pair provided in this kit recognizes human and mouse 4E-BP1 phosphorylated at site Threonine-36 and total 4E-BP1.
RayBio® Phospho-4E-BP1 (Thr36) and Total 4E-BP1 ELISA kit is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in human and mouse cell lysates. By determining 4E-BP1 protein in your experimental model system, you can verify pathway activation in your cell lysates. You can simultaneously measure numerous different cell lysates without spending excess time and effort in performing a Western Blotting analysis.
This Sandwich ELISA kit is an in vitro enzyme-linked immunosorbent assay for the measurement of human and mouse phospho-4E-BP1 and total 4E-BP1. An anti-pan 4E-BP1 antibody has been coated onto a 96-well plate. Samples are pipetted into the wells and 4E-BP1 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and rabbit anti-4E-BP1 (Thr36) antibody is used to detect phosphorylated 4E-BP1 or biotinylated anti-4E-BP1 antibody is used to detect pan 4E-BP1. After washing away unbound antibody, HRP-conjugated anti-rabbit IgG or HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of 4E-BP1 (Thr36) or pan 4E-BP1 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.
Product Features
Simultaneously measure phosphorylated protein and pan protein in one experiment (for normalization purpose)
Screen numerous different cell lysates without performing a Western Blot analysis
Minimal hands-on time, convenient, and non-radioactive material
Application Notes
Kit Components
Pre-Coated 96-well Strip Microplate
Wash Buffer
Anti-Phospho Antibody
Anti-Pan Antibody
HRP-Conjugated Secondary Antibody
Streptavidin-Conjugated HRP
Assay Diluent
TMB One-Step Substrate
Stop Solution
Lysis Buffer
Positive Control Sample
Other Materials Required
Distilled or deionized water
100 ml and 1 liter graduated cylinders
Tubes to prepare sample dilutions
Protease and Phosphatase inhibitors
Precision pipettes to deliver 2 µl to 1 ml volumes
Adjustable 1-25 ml pipettes for reagent preparation
Benchtop rocker or shaker
Microplate reader capable of measuring absorbance at 450 nm
Protocol Outline
Prepare all reagents and samples as instructed in the manual.
Add 100 µl of sample or positive control to each well.
Incubate 2.5 h at RT or O/N at 4 °C.
Add 100 µl of prepared primary antibody to each well.
Incubate 1 h at RT.
Add 100 µl of prepared 1X HRP-Streptavidin to each well.
Incubate 1 h at RT.
Add 100 µl of TMB One-Step Substrate Reagent to each well.
Incubate 30 min at RT.
Add 50 µl of Stop Solution to each well.
Read at 450 nm immediately.
Typical Data
Positive Control
HeLa cells were treated with FBS. Solubilize cells at 4 x 107 cells/ml in Cell Lysate Buffer. Serial dilutions of lysates were analyzed in this ELISA. Please see step 3 of Part VI Reagent Preparation for detail.
Insulin Stimulation of 293 Cell Line
293 cells were treated or untreated with Insulin. Cell lysates were analyzed using this phosphoELISA and Western Blot.
Storage/Stability
Upon receipt, the kit should be stored at –20°C. Please use within 6 months from the date of shipment. After initial use, Wash Buffer Concentrate (Item B), Assay Diluent (Item E), TMB One-Step Substrate Reagent (Item H), Stop Solution (Item I) and Cell Lysate Buffer (Item J) should be stored at 4°C to avoid repeated freeze-thaw cycles. Return unused wells to the pouch containing desiccant pack, reseal along entire edge, and store at –20°C. Item D, store at 2-8°C for up to one month (store at -20°C for up to 6 months, avoid repeated freeze-thaw cycles). Reconstituted Positive Control (Item K) should be stored at -70°C.
I used this ELISA with mouse tissue lysate where the mice were injected with -/+ insulin. Don't forget to take into account extra wells to test the optimal protein concentration of lysate to use. I found myself needing more wells than originally anticipated. Otherwise, a very straightforward kit to use.
