Description
LAT (Phospho-Tyr191)Colorimetric Cell-Based ELISA Kit
The LAT Phospho-Tyr191 Colorimetric Cell-Based ELISA Kit is a cutting-edge assay designed for the detection and quantification of phosphorylated LAT (Linker for Activation of T cells) at tyrosine 191 in cell lysates. This kit offers high sensitivity and specificity, ensuring accurate and reliable results for your research needs.LAT is a crucial adaptor protein in T cell activation, playing a key role in signal transduction pathways that regulate immune responses. Phosphorylation of LAT at tyrosine 191 is a critical event in T cell receptor signaling, making this ELISA kit essential for studying T cell activation and immune system function.
With its easy-to-use protocol and robust performance, the LAT Phospho-Tyr191 Colorimetric Cell-Based ELISA Kit is ideal for a wide range of applications in immunology, cell biology, and drug development. Enhance your research with this advanced assay and uncover new insights into T cell signaling pathways.
| Product Name: | LAT (Phospho-Tyr191)Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB01299 |
| ELISA Type: | Cell-Based |
| Target: | LAT (Phospho-Tyr191) |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The LAT (Phospho-Tyr191) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect LAT protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated LAT in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on LAT phosphorylation.
Qualitative determination of LAT (Phospho-Tyr191) concentration is achieved by an indirect ELISA format. In essence, LAT (Phospho-Tyr191) is captured by LAT (Phospho-Tyr191)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:
| 1. | A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values. |
| 2. | Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted. |
| Database Information: | Gene ID: 27040, UniProt ID: O43561, OMIM: 602354, Unigene: Hs.632179 |
| Gene Symbol: | LAT |
| Sub Type: | Phospho |
| UniProt Protein Function: | Required for TCR (T-cell antigen receptor)- and pre-TCR-mediated signaling, both in mature T-cells and during their development. Involved in FCGR3 (low affinity immunoglobulin gamma Fc region receptor III)-mediated signaling in natural killer cells and FCER1 (high affinity immunoglobulin epsilon receptor)-mediated signaling in mast cells. Couples activation of these receptors and their associated kinases with distal intracellular events such as mobilization of intracellular calcium stores, PKC activation, MAPK activation or cytoskeletal reorganization through the recruitment of PLCG1, GRB2, GRAP2, and other signaling molecules. |
| NCBI Summary: | The protein encoded by this gene is phosphorylated by ZAP-70/Syk protein tyrosine kinases following activation of the T-cell antigen receptor (TCR) signal transduction pathway. This transmembrane protein localizes to lipid rafts and acts as a docking site for SH2 domain-containing proteins. Upon phosphorylation, this protein recruits multiple adaptor proteins and downstream signaling molecules into multimolecular signaling complexes located near the site of TCR engagement. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Jul 2008] |
| UniProt Code: | O43561 |
| NCBI GenInfo Identifier: | 14194891 |
| NCBI Gene ID: | 27040 |
| NCBI Accession: | O43561.1 |
| UniProt Secondary Accession: | O43561,O43919, B7WPI0, C7C5T6, G5E9K3, |
| UniProt Related Accession: | O43561 |
| Molecular Weight: | 27,774 Da |
| NCBI Full Name: | Linker for activation of T-cells family member 1 |
| NCBI Synonym Full Names: | linker for activation of T-cells |
| NCBI Official Symbol: | LAT |
| NCBI Official Synonym Symbols: | LAT1; pp36 |
| NCBI Protein Information: | linker for activation of T-cells family member 1 |
| UniProt Protein Name: | Linker for activation of T-cells family member 1 |
| UniProt Synonym Protein Names: | 36 kDa phospho-tyrosine adapter protein; pp36; p36-38 |
| Protein Family: | Latexin |
| UniProt Gene Name: | LAT |
| UniProt Entry Name: | LAT_HUMAN |
| Component | Quantity |
| 96-Well Cell Culture Clear-Bottom Microplate | 2 plates |
| 10X TBS | 24 mL |
| Quenching Buffer | 24 mL |
| Blocking Buffer | 50 mL |
| 15X Wash Buffer | 50 mL |
| Primary Antibody Diluent | 12 mL |
| 100x Anti-Phospho Target Antibody | 60 µL |
| 100x Anti-Target Antibody | 60 µL |
| Anti-GAPDH Antibody | 60 µL |
| HRP-Conjugated Anti-Rabbit IgG Antibody | 12 mL |
| HRP-Conjugated Anti-Mouse IgG Antibody | 12 mL |
| SDS Solution | 12 mL |
| Stop Solution | 24 mL |
| Ready-to-Use Substrate | 12 mL |
| Crystal Violet Solution | 12 mL |
| Adhesive Plate Seals | 2 seals |
The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:
- Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
- Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
- 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
- Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
- Graph paper or computer software capable of generating or displaying logarithmic functions
- Absorbent papers or vacuum aspirator
- Test tubes or microfuge tubes capable of storing ≥1 ml
- Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
- Orbital shaker (optional)
- Deionized or sterile water
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1. | Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37 °C prior to adding cells. |
| 2. | Incubate the cells for overnight at 37 °C, 5% CO2. |
| 3. | Treat the cells as desired. |
| 4. | Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice. |
| 5. | Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells. |
| 6. | Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4 °C for a week. |
| 7. | Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature. |
| 8. | Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time. |
| 9. | Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature. |
| 10. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 11. | Add 50 µL of 1x primary antibodies Anti-LAT (Phospho-Tyr191) Antibody, Anti-LAT Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4 °C. If the target expression is known to be high, incubate for 2 hours at room temperature. |
| 12. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 13. | Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature. |
| 14. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 15. | Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark. |
| 16. | Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader. |
(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)
