Proteins were extracted with lysis buffer (50mM HEPES, pH 7.4, 80mM NaCl, 5mM MgCl2, 10mM EDTA pH 8.0, 5mM Sodium Pyrophosphate, 1% Triton X-100) and protease inhibitor cocktail (Sigma-Aldrich). Fifty μg of protein was resolved on a 4-20% SDS polyacrylamide gel (Invitrogen), transferred onto nitrocellulose membranes (Bio-Rad), and analyzed by immunoblotting using anti-NFAT5 (Thermo Scientific), anti-NFAT1 (Thermo Scientific), and anti-β-actin (Sigma-Aldrich) primary antibodies, followed by anti-mouse IRdye 800 and anti-rabbit DyLight 680-conjugated secondary antibodies (Rockland).
Anti nfat1
Manufactured by Thermo Fisher Scientific
Anti-NFAT1 is a laboratory reagent used to detect and quantify the presence of the NFAT1 protein in biological samples. It functions as a specific antibody that binds to the NFAT1 protein, allowing for its identification and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Sds polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Anti nfat5, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Formaldehyde, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
2 protocols using anti nfat1
1
Immunoblotting Analysis of NFAT Proteins
2
Immunofluorescence Analysis of NFAT1 in T Cells
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After stimulation, primary T cells or Jurkat cells were fixed with 4% formaldehyde (Affymetrix) at room temperature for 15 minutes, air-dried on superfrost microscopic glass slides, and blocked with 5% normal goat serum before being incubated overnight with primary antibodies: mouse monoclonal Alexa 647-conjugated anti-HA (6E2, Cell Signaling Technology), anti-NFAT1 (MA1-025, Thermo Scientific), or rabbit polyclonal pS79-NFAT1. Species-matched secondary antibodies conjugated with fluorochrome dye Alexa 594 or Alexa 488 were added for 1 hour. The slides were washed, air-dried, and mounted directly by Prolong Diamond Antifade Mountant with DAPI (P36962, Molecular Probes). Cells stained with only secondary antibodies were used as controls. Images were taken with a Zeiss confocal microscope (Nikon Instruments, Melville, New York, United States), and 10–15 fields/condition were analyzed with ImageJ. PLA data were analyzed by Image-Pro Premier program (version 9.3.1).
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