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PSAT1 is a laboratory equipment product from Thermo Fisher Scientific. It is designed to perform protein stability assays. The core function of the PSAT1 is to measure the thermal stability of proteins in a high-throughput manner.

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Lab products found in correlation

Gsk 3β, by Thermo Fisher Scientific (1 mentions) Anti gapdh antibody, by Thermo Fisher Scientific (1 mentions) E cadherin, by Thermo Fisher Scientific (1 mentions) Vimentin, by Thermo Fisher Scientific (1 mentions) Ripa buffer, by Cell Signaling Technology (1 mentions) Snail, by Thermo Fisher Scientific (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) 7500 software v2, by Thermo Fisher Scientific (1 mentions) Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions) Taqman master mix, by Thermo Fisher Scientific (1 mentions) Actb β actin, by Thermo Fisher Scientific (1 mentions) Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (1 mentions) Quantstudio design analysis software, by Thermo Fisher Scientific (1 mentions) Phgdh, by Thermo Fisher Scientific (1 mentions) Omniscript rt kit, by Qiagen (1 mentions) Protein assay dye reagent, by Bio-Rad (1 mentions) Protease inhibitor, by Roche (1 mentions) Amersham hyperfilm ecl, by GE Healthcare (1 mentions) Anti hsc70, by Santa Cruz Biotechnology (1 mentions) Curix 60 developer, by AGFA HealthCare (1 mentions)

Spelling variants of this product

Anti-PSAT1 (2 citations)

4 protocols using psat1

1

Western Blot Analysis of Cellular Proteins

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After receiving the treatment, the cells were subjected to lysis using RIPA buffer (Cell Signaling Tech, Inc, Danvers, MA, USA), and the extracts were submitted to sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by transfer to nitrocellulose membranes and blotting. The membranes were blocked using a mixture of 5% skim milk and 0.1% Tween 20 in Tris-buffered saline. The membranes were incubated with Iry antibodies at 4°C for 12 h. PSAT1, p-GSK-3 β, Snail, E-cadherin, GSK-3β, Vimentin (Thermo Fisher Scientific), and the anti-GAPDH antibody were used as internal controls (Thermo Fisher Scientific). The blots were visualized using enhanced chemiluminescence after incubating with secondary antibodies for 2 h.
Sun C., Zhang X., Chen Y., Jia Q., Yang J, & Shu Y. (2018). MicroRNA-365 suppresses cell growth and invasion in esophageal squamous cell carcinoma by modulating phosphoserine aminotransferase 1. Cancer Management and Research, 10, 4581-4590.
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2

Quantitative Analysis of mRNA Expression

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mRNA levels were assessed using quantitative real-time PCR (qRT-PCR). Total RNA was extracted from cells usingthe RNeasy Mini Kit (Qiagen) as per the manufacturer’s protocol. RNA yield was quantified using the NanoDrop ND-1000 (ThermoFisher Scientific) and 1 mg of RNA was reverse transcribed to cDNA using the Omniscript RT Kit (Qiagen). qPCR was performed using the TaqMan™ assay system.
The following TaqMan™gene expression probes were used:PHGDH (Hs00198333_m1, ThermoFisher Scientific); PSAT1 (Hs00795278_mH, ThermoFisher Scientific); PSPH (Hs00190154_m1, ThermoFisher Scientific); ACTB (β-Actin, 4310881E, Applied Biosystems).
Assay mixtures were prepared consisting of 10 μl TaqMan™ Master Mix (Applied Biosystems), 1 μl TaqMan™ gene probe & 1 μl cDNA, topped up to 20 μl with 8μl RNase free H2O. The qPCR reaction was carried out using either the 7500 Real Time or the QuantStudio 5 Real Time PCR systems (Applied Biosystems) and the process was 2 minutes at 50ºC, followed by 10 minutes holding at 95ºC, then 40 cycles of 15 seconds at 95ºC and 1 minute at 60ºC. Relative mRNA quantifications were obtained using the comparative Ct method, and data was analysed using either the 7500 software v2.3 or QuantStudio Design & Analysis Software (Applied Biosystems).
Xu R., Jones W., Wilcz‐Villega E., Costa A.S., Rajeeve V., Bentham R.B., Bryson K., Nagano A., Yaman B., Olendo Barasa S., Wang Y., Chelala C., Cutillas P., Szabadkai G., Frezza C, & Bianchi K. (2020). The breast cancer oncogene IKKε coordinates mitochondrial function and serine metabolism. EMBO Reports, 21(9), e48260.
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3

Quantitative Immunoblotting for Serine Biosynthesis Enzymes

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Cells were lysed on ice for 30 minutes using lysis buffer (1% [vol/vol] Nonidet P-40, 20 mM Tris-HCl [pH 8.0], 150 mM NaCl, and 5 mM EDTA with protease inhibitors [Roche Diagnostics, 11873580001]) and phosphatase inhibitors (sodium fluoride and sodium orthovanadate;MilliporeSigma, S7920 and S6508). Samples were centrifuged (21,000g, 15 minutes, 4°C), and the protein content of the supernatant was measured using the Protein Assay Dye Reagent (Bio-Rad, 500-0006). Immunoblotting was performed using from 10 μg to 50 μg of protein lysate. Antibodies were used for detecting the following proteins: PHGDH (Cell Signaling Technology; human specific, 66350; mouse-specific, 13428), PSAT1 (Thermo Fisher Scientific, PA5-22124), PSPH (Thermo Fisher Scientific, PA5-22003), anti-HSC70 (Santa Cruz Biotechnology, sc-7298). All secondary HRP-conjugated antibodies were from Cell Signaling Technology. Membranes were exposed using Amersham Hyperfilm ECL (GE Healthcare) and developed using a Curix 60 developer (Agfa). Films were scanned and quantified using ImageJ 1.50c (NIH). All values were normalized to the HSC70 loading control, and relative fold-change was calculated with the isotype control antibody treated cells taken as 100% of expression.
D’Avola A., Legrave N., Tajan M., Chakravarty P., Shearer R.L., King H.W., Kluckova K., Cheung E.C., Clear A.J., Gunawan A.S., Zhang L., James L.K., MacRae J.I., Gribben J.G., Calado D.P., Vousden K.H, & Riches J.C. (2022). PHGDH is required for germinal center formation and is a therapeutic target in MYC-driven lymphoma. The Journal of Clinical Investigation, 132(9), e153436.
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4

Western Blot Analysis of Metabolic Enzymes

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Cells were lysed in mammalian cell lysis buffer (50mM Tris pH 7.5, 150mM NaCl and 0.5% NP40) containing a protease and phosphatase inhibitor cocktail (Bimake.com) and 1 µM MG132 (Selleckchem). Protein concentration was determined by BCA assay (Thermo Fisher). Quantified protein samples were separated by electrophoresis on 4-20% readymade Tris-Glycine gels (Invitrogen) and transferred to PVDF membranes (Millipore).
Membranes were blocked with 2% bovine serum albumin for 1 h and incubated overnight with one or more primary antibody: PSAT1 (Thermo Fisher, PA5-22124.), PHGDH (Sigma, HPA021241), PSPH (Santa Cruz, sc-365183), and Actin (Sigma, A1978). Overexpression and knockout of PSAT1 (Figure 3) confirmed the correct band on PSAT1 western blots.
Membranes were washed with tween 20-containing tris buffered saline and incubated with fluorescence-conjugated secondary antibodies (Bio-Rad). Rhodamine-conjugated anti-tubulin was also treated as a secondary antibody (Bio-Rad, 12004166). Images were detected using a ChemiDoc MP Imaging System (Bio-Rad).
Choi B., Conger K.O., Selfors L.M., & Coloff J.L. (2020). Lineage-Specific Silencing of PSAT1 Induces Serine Auxotrophy and Sensitivity to Dietary Serine Starvation in Luminal Breast Tumors.
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