Lat py226

Manufactured by BD

The LAT pY226 is a laboratory equipment product. It is designed for specific laboratory functions. Details regarding its core function and intended use are not available in a concise, unbiased, and factual manner.

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Lab products found in correlation

Slp 76 py128, by BD (2 mentions) Sea block buffer, by Thermo Fisher Scientific (2 mentions) Pvdf membrane, by Merck Group (2 mentions) Plcγ1, by Cell Signaling Technology (1 mentions) Src py416, by Cell Signaling Technology (1 mentions) Grb2 clone 23, by Santa Cruz Biotechnology (1 mentions) Py783 plc γ1, by Cell Signaling Technology (1 mentions) Actin clone c4, by Merck Group (1 mentions) Criterion polyacrylamide gel, by Bio-Rad (1 mentions) P38 pt180 py182, by Cell Signaling Technology (1 mentions) Pvdf membrane, by Santa Cruz Biotechnology (1 mentions) Sea block, by Thermo Fisher Scientific (1 mentions) Criterion gel, by Bio-Rad (1 mentions) Pvdf fl membrane, by Merck Group (1 mentions) Gapdh, by Meridian Bioscience (1 mentions) Plc γ py783, by Cell Signaling Technology (1 mentions) Am tirf mc system, by Leica camera (1 mentions) Alexa fluor 488 goat anti mouse igg1, by Thermo Fisher Scientific (1 mentions) Total akt, by Cell Signaling Technology (1 mentions)

4 protocols using lat py226

Quantitative Immunoblotting of Cell Signaling

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Protein containing samples (equivalent of 5 × 10⁶ cells) were loaded onto a 4–15% precast Criterion polyacrylamide gel (Biorad). The separated proteins were transferred onto PVDF membranes (Millipore), and then blocked for 1 h at room temperature in a 1:1 1XPBS:SEA Block buffer (Thermo Scientific). The PVDF membranes were then incubated with primary antibodies against GRB2 (clone 23, Santa Cruz Biotechnology), LAT pY226 (clone J96-1238.58.93, BD Pharmingen), LAT pY132 (Genetex), SLP-76 pY128 (clone J141-668.36.58, BD Pharmingen), ERK1/ERK2 pY187/pT185 (Invitrogen), p38 pT180/pY182 (Cell Signaling), JNK pT183/pY195 (Cell Signaling), Akt pS473 (clone-14-6, Invitrogen), Src pY416 (Cell Signaling), pY783 PLC-γ1 (Cell signaling), PLC-γ1 (Cell Signaling), lymphocyte-specific protein tyrosine kinase (LCK) (Cell Signaling), pY (4G10, Millipore), Gsk3αβ pS21/9 (Cell Signaling), actin (clone C4, Millipore), or GAPDH (Meridian Life Sciences). Secondary antibodies conjugated to IRDye 800CW or IRDye 680 were diluted in SEA Block and incubated with the PVDF membranes for 30 min at room temperature. The membranes were then visualized using the Licor Odyssey Infrared detector.

Bilal M.Y, & Houtman J.C. (2015). GRB2 Nucleates T Cell Receptor-Mediated LAT Clusters That Control PLC-γ1 Activation and Cytokine Production. Frontiers in Immunology, 6, 141.

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Western Blotting of Phospho-Proteins

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Cellular lysates were separated by polyacrylamide gel electrophoresis (SDS-PAGE) using 4-15% Criterion gels (Biorad) and transferred to PVDF-FL membrane (Millipore). To analyze site-specific phosphorylation the membranes were blocked for 1 hr at room temperature in a blocking buffer that was 1:1 1x PBS:SEA Block (Thermo Scientific). The PVDF membranes were incubated with primary antibodies against LAT pY132 (Biosource), LAT pY226 (BD Pharmingen), ZAP-70 pY319 (Cell Signaling Technologies), SLP-76 pY128 (BD Pharmingen), PLC-γ1 pY783 (Cell Signaling Technologies), ERK1/ERK2 pT185/pY187 (Thermo Scientific) and GAPDH (Meridian Life Sciences). IRDye 800CW or IRDye 680-conjugated secondary antibodies were diluted in SEA Block as above and incubated with the PVDF membrane for 30 min at room temperature. The membranes were then imaged using the Licor Odyssey. The intensity of the immunoblotting bands was determined using Odyssey’s v3.0 software. Phosphorylated proteins were normalized to GAPDH and the relative amount of phosphorylation to the 2 minute (for LAT pY132, LAT pY226, ZAP-70, SLP-76 and PLC-γ1) or 5 minute (ERK1/ERK2) LAT WT bands were determined as described previously (35 (link)-37 (link)). The data from 4-5 independent replicates were compiled and then plotted using Prism (GraphPad Software).

Tremblay M.M., Ollinger T, & Houtman J.C. (2020). The Membrane Proximal Proline-Rich Region and Correct Order of C-Terminal Tyrosines on the Adaptor Protein LAT Are Required For TCR-Mediated Signaling and Downstream Functions. Cellular signalling, 76, 109790.

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Western Blot Analysis of Signaling Proteins

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Following SDS-PAGE, resolved proteins were transferred onto a PVDF membrane (Millipore) and then blocked for 1 h at room temperature in a 1:1 solution of SEA Block buffer (Thermo Scientific) in 1X PBS. Membranes were incubated for 1 h at room temperature or overnight at 4 °C with primary antibodies followed by two washes in 0.05% Tween-20 in 1X PBS. Secondary anti-mouse or anti-rabbit DyLight 680- or 800-conjugated antibodies were applied for 1 h at room temperature followed by 2 washes. Blots were then visualized using the Licor Odyssey Infrared detector. Densitometric analysis of protein bands was performed using Odyssey’s v3.0 software and normalized to GAPDH. The followed primary antibodies were used: GRB2 (BD Pharmingen or Cell Signaling Technology); LAT pY226 (clone J96-1238.58.93, BD Pharmingen); and GAPDH (Meridian Bioscience).

Sandouk A., Xu Z., Baruah S., Tremblay M., Hopkins J.B., Chakravarthy S., Gakhar L., Schnicker N.J, & Houtman J.C. (2023). GRB2 dimerization mediated by SH2 domain-swapping is critical for T cell signaling and cytokine production. Scientific Reports, 13, 3505.

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Imaging T-Cell Receptor Signaling

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APBTs were stimulated in the presence of 0.1% ethanol control or 10 μg/ml GML with plate-bound anti-CD3 on glass coverslips, fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton-X, and stained as described below. To detect microcluster formation of TCR signaling proteins, antibodies specific for LAT pY226 (BD Pharmingen), PLC-γ pY783 (Cell Signaling, and total AKT (Cell signaling) were incubated with fixed and permeabilized cells overnight for 4°C. Subsequently, conjugated secondary antibodies Dylight Goat anti-rabbit IgG and DyLight 568 Goat anti-rabbit IgG (Biolegend) as well as Alexa Fluor 488 goat anti-mouse IgG1 (Thermo Fisher) were incubated with the cells at room temperature for 2 hours. All images were captured by the Leica AM TIRF MC system using 100x magnification oil immersion objective lens at room temperature at the University of Iowa Central Microscopy Research Facility.

Zhang M.S, & Houtman J.C. (2016). Human Serum Albumin (HSA) Suppresses the Effects of Glycerol Monolaurate (GML) on Human T Cell Activation and Function. PLoS ONE, 11(10), e0165083.

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