ArtNr |
PEL-LCK-Y394-T-5 |
Hersteller |
Raybiotech
|
Menge |
5 Plate Kit |
Quantity options |
1 Plate Kit
2 Plate Kit
5 Plate Kit
|
Kategorie |
|
Typ |
Elisa-Kit |
Specific against |
other |
ECLASS 10.1 |
32160605 |
ECLASS 11.0 |
32160605 |
UNSPSC |
41116126 |
Lieferbar |
|
Specificity |
The antibody pair provided in this kit recognizes human LCK phosphorylated at Tyrosine-394 and total LCK. |
Species Detected |
Human |
Compatible Sample Types |
Cell Lysates Tissue Lysates |
Design Principle |
Sandwich-based |
Method of Detection |
Colorimetric |
Quantitative/Semi-Quantitative |
Semi-Quantitative |
Solid Support |
96-well Microplate |
Gene Symbols |
LCK |
Kit Components |
Pre- Coated 96-well Strip Microplate Wash Buffer Anti- Phospho Antibody Anti- Pan Antibody H R P- Conjugated Secondary Antibody Streptavidin- Conjugated H R Pli id=""> Assay Diluent T M B One- Step Substrate Stop Solution Lysis Buffer Positive Control Sample |
Other Materials Required |
Distilled or deionized water100 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 R T or O/ N at 4 ° C. Add 100 µl of prepared primary antibody to each well. Incubate 1 h at R T. Add 100 µl of prepared 1 X H R P- Streptavidin to each well. Incubate 1 h at R T. Add 100 µl of T M B One- Step Substrate Reagent to each well. Incubate 30 min at R T. Add 50 µl of Stop Solution to each well. Read at 450 nm immediately. |
Protein Name & Synonyms |
Tyrosine-protein kinase Lck (EC 2.7.10.2) (Leukocyte C-terminal Src kinase) (LSK) (Lymphocyte cell-specific protein-tyrosine kinase) (Protein YT16) (Proto-oncogene Lck) (T cell-specific protein-tyrosine kinase) (p56-LCK) |
Pathway |
T Cell ReceptorTyrosine Kinase Family |
Introduction |
RayBio® Phospho-LCK (Y394) and Total LCK ELISA kit is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in human cell lysates. By determining phosphorylated LCK 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 phospho-LCK and total LCK. An anti-pan LCK antibody has been coated onto a 96-well plate. Samples are pipetted into the wells and LCK present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-LCK (Y394) antibody is used to detect phosphorylated LCK or biotinylated anti-LCK antibody is used to detect pan LCK. After washing away unbound antibody, 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 LCK (Y394) or pan LCK bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. |
Positive Control |
JURKAT cells were treated with Pervanadate at 37°C for 10 min. 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. |
Pervanadate Stimulation of JURKAT Cell Lines |
JURKAT cells were treated or untreated with Pervanadate for 10 min. Cell lysates were analyzed using this phosphoELISA and Western Blot. |
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