About 1 × 107 CD4+ T-cells were stimulated with aCD3-/CD28-coated microbeads for 30 min in the presence/absence of PRBCs or 1 mM NAC. Thereafter, cells were washed thoroughly using RBC lysis buffer (154 mM NH4Cl, 10 mM KHCO3, and 100 μM Na2 EDTA). Washed CD4+ T-cells were lysed with radioimmunoprecipitation assay buffer supplemented with protease inhibitor and phosphatase inhibitors (all Sigma–Aldrich). After 30 min of incubation on ice with periodic pulse vortexing, cell lysates were centrifuged at 16,000g for 15 min at +4 °C. For equal loading, protein concentration was measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to manufacturing instructions. A 4%–12% SDS-PAGE (BioRad) was used to separate proteins following a transfer onto polyvinylidene difluoride membranes (GE Healthcare). The membrane was blocked with bovine serum albumin (Sigma–Aldrich), and the following antibodies were used for incubation overnight: Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (1:2000, #2701; Cell Signaling Technology) and total Zap-70 (1:1000, 99F2; Cell Signaling Technology). Protein bands were visualized using horseradish peroxidase–linked anti-rabbit (Cell Signaling Technology) or antigoat (Abcam) antibodies and SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).
Total zap 70
Manufactured by Cell Signaling Technology
Total ZAP-70 is a cell-based ELISA kit designed to quantify the total expression of the ZAP-70 protein. ZAP-70 is a key signaling molecule involved in T-cell receptor signaling. The kit provides a reproducible and sensitive method to measure the total levels of ZAP-70 in cell lysates.
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Lab products found in correlation
Iblot transfer system, by Thermo Fisher Scientific (2 mentions)
Supersignal west dura, by Thermo Fisher Scientific (2 mentions)
Plat y226, by BioLegend (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor and phosphatase inhibitor, by Merck Group (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Phospho zap 70 tyr319 syk tyr352, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by GE Healthcare (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Radioimmunoprecipitation assay buffer, by Merck Group (1 mentions)
Anti ha, by Cell Signaling Technology (1 mentions)
Pzap 70, by Cell Signaling Technology (1 mentions)
P lck, by R&D Systems (1 mentions)
Plck y394, by R&D Systems (1 mentions)
Gapdh and β actin, by Merck Group (1 mentions)
Total lck, by Cell Signaling Technology (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Human il 2 elisa kit, by BD (1 mentions)
4 protocols using total zap 70
1
Phospho-Zap-70 Immunoblotting Protocol
2
Immunoblot Analysis of Signaling Proteins
3
Western Blot Analysis of Signaling Proteins
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Cellular lysates were mixed with Laemmli sample buffer, heated at 95 °C for 5 min and separated on NuPAGE Bis-Tris gels by electrophoresis and transferred to nitrocellulose membranes using the iBlot transfer system (Thermo Scientific). Membranes were incubated in previously heated 3% fat-free dry milk for 1 h at room temperature, followed by overnight incubation with primary antibodies. Proteins were detected with Super Signal West Dura (Thermo Scientific, #34075) using a Bio-Rad ChemiDoc MP imaging system. Immunoblots were quantified using ImageJ (NIH) by stripping and reprobing the same membrane for the indicated loading controls for normalization. Integrated Density (IntDen, the product of area and mean gray value) was calculated for each condition. In each experiment, the IntDen value was obtained for each condition and was normalized to the value obtained in the control stimulated LV condition using the following formula: (IntDen value/IntDen value at stimulated LV). Primary antibodies used were: pLck (Y394, R&D Systems), pZAP-70 (Y319), total Lck, total ZAP-70, total LAT and anti-HA from Cell Signaling, pLAT(Y226) from BioLegend, and β-actin and GAPDH from Sigma.
4
Quantification of IL-2 Cytokine Release
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IL-2 cytokine released into cell culture supernatant was quantified using human IL-2 ELISA kit (BD Biosciences) according to the manufacturer’s instructions. Jurkat cells were stimulated with anti-CD3 (5 μg/ml) for the indicated times prior to addition of cell lysis buffer (Cell Signaling). Following PMA/Ionomycin stimulation for 15 minutes, nuclear proteins were isolated using nuclear protein isolation kit (NEPER, Thermo Scientific) following manufacturer’s instructions. Proteins were separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (BIORAD). Membranes were incubated in protein-free blocking buffer (Thermo Scientific) for 1 hour at room temperature followed by incubation with primary antibodies. Proteins were detected with Amersham ECL (GE Healthcare) using a Kodak Imager. Primary antibodies used were: NFAT and pLAT(Y226; BD Biosciences); total LAT (Biolegend); pZAP70 (Y319); total ZAP70; pLck (Y394/ pSrcY416); total Lck (Y394); Csk; YY1 (all from Cell Signaling Technology); PTPRE (Origene, clone 4B2). Immunoblots were quantified by ImageJ.
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