20 µg of total protein was loaded onto a 10% SDS-PAGE gel (Cat: #1610158, Bio-Rad, USA) and blotted to Nitro cellulose membranes (Cat: 88018, Thermo Fisher Scientific, the Netherlands) followed by incubation with block buffer (Cat: P/N 927-40000, Licor, Bioscience, USA). Protein levels were assessed by immunoblotting using specific antibodies against LAT2 (Cat: 11986S, Cell Signaling Technology, Leiden, the Netherlands, 1:1000), and β-actin (Cat: ab8229, Abcam, Cambridge, UK, 1:500) as a loading control, followed by incubation with secondary antibodies (IRdye 680 CW, Cat: P/N 925-68071, P/N 925-68074; IRDye 800 CW, Cat: P/N 925-32211, P/N 925-32214, Licor Bioscience, USA) and detection of signals using the Odyssey imaging system (Licor Bioscience, USA).
Block buffer
Manufactured by LI COR
Sourced in Netherlands
Block buffer is a solution used to prevent non-specific binding of proteins in immunological assays. It contains a combination of proteins, detergents, and other additives that help to minimize background signal and improve the specificity of the assay.
Automatically generated - may contain errors
Lab products found in correlation
Sds page gel, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
Irdye 680cw, by LI COR (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Anti ripk1, by Cell Signaling Technology (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Protease phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Anti p ripk1, by Cell Signaling Technology (1 mentions)
Ab46545, by Abcam (1 mentions)
2 protocols using block buffer
1
Quantification of LAT2 Protein Expression
2
Immunoblot Analysis of Oxidative Stress Markers
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Cells were lysed in RIPA buffer (Thermo Scientific, 78430) with protease & phosphatase inhibitor cocktail (Thermo Scientific, 78441). Equal amount of total protein (35 μg/well) was boiled in 1x Laemmli sample buffer (with 1/10 vol β-mercapto-ethanol) and loaded onto SDS-PAGE gel. After resolution by electrophoresis, proteins were transferred to nitrocellulose membrane (Bio-Rad, 1620115) in ice bath with stirring and treated in block buffer (LI-COR) for 1 h. Membranes were incubated with primary antibody (1:1000) overnight at 4 °C followed by secondary antibodies (1:5000) incubation for 1 h at room temperature and then were imaged. Primary antibodies: anti-pRIPK1: Cell Signaling, 65746S, anti-RIPK1: Cell Signaling, 3493S, anti-4-HNE modified proteins: Abcam, ab46545, anti-GPX4: Abcam, ab231174; anti-α-Tubulin: Sigma, 05-829. Secondary antibodies: LI-COR 680RD goat anti-rabbit IgG (LI-COR, 926-68071), LI-COR 800RD goat anti-mouse IgG (LI-COR, 926-32210).
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