eIF4EBP1 (EIF4EBP1)
(Eukaryotic Translation Initiation Factor 4E Binding Protein 1 (EIF4EBP1))
Binding Specificity
pThr36, total
Reactivity
Mouse, Human
Detection Method
Colorimetric
Method Type
Sandwich ELISA
Application
ELISA
Purpose
Human/Mouse Phospho-4E-BP1 (T36) and Total 4E-BP1 ELISA Kit. This assay semi-quantitatively measures phophorylated 4E-BP1 (Thr36) and Total 4E-BP1 in lysate samples.
Sample Type
Cell Lysate, Tissue Lysate
Analytical Method
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.
Characteristics
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
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
Material not included
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
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.
Restrictions
For Research Use only
Storage
-20 °C
Storage Comment
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), HRP-Streptavidin (Item G), 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. Reconstituted Positive Control (Item K) should be stored at -70 °C.