Cells were lysed in ice cold radioimmunoprecipitation buffer (50 mM Tris–HCl, pH 8, 0.1% SDS, 150 mM NaCl, 0.25% deoxycholic acid, 1 mM EDTA, and 1% NP-40) supplemented with 1:500 (v/v) protease inhibitor cocktail (Sigma). Lysates were centrifuged (13,000 rpm for 10 min at 4 °C) and the cleared lysates were diluted with Laemmli buffer (Bio-Rad). Lysates were resolved on a 4 to 15% gradient SDS-PAGE gel (Bio-Rad), transferred onto a nitrocellulose membrane (Bio-Rad), and probed with the indicated primary antibodies and corresponding secondary antibodies. Proteins were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and a Bio-Rad ChemiDoc MP Imaging System. Primary antibodies used in this study in the order of appearance are as follows: SAMHD1 (abcam; 67820), GAPDH (Cell Signaling Technology; 14C10), RNR M1 (abcam; 137114), RNR M2 (abcam; 172476), thymidine kinase 1 (abcam; 76495), dCK (abcam; 96599), nucleoside diphosphate kinase (abcam; 241162), p-SAMHD1 (Cell Signaling Technology; 89930S), PP2A B55α (Cell Signaling Technology; 4953T), CDK1 (abcam; 133327), cyclin A2 (abcam; 181591), E2F-1 (Cell Signaling Technology; 3742S), and CDK2 (abcam; 32147). Secondary antibodies used in this study are as follows: anti-mouse (Cytiva; NA931V) and anti-rabbit (Cytiva; NA934V).
4 to 15 gradient sds page gel
Manufactured by Bio-Rad
The 4 to 15% gradient SDS-PAGE gel is a laboratory equipment used for the separation and analysis of proteins based on their molecular weight. It consists of a polyacrylamide gel with a linear gradient of 4 to 15% acrylamide concentration, which allows for the effective separation of a wide range of protein sizes.
Automatically generated - may contain errors
Lab products found in correlation
Cyclin a2, by Abcam (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Samhd1, by Abcam (1 mentions)
Psamhd1, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Gena931, by Merck Group (1 mentions)
Goat anti mouse secondary antibody, by Merck Group (1 mentions)
2 protocols using 4 to 15 gradient sds page gel
1
Western Blot Analysis of Cell Lysates
2
SARS-CoV-2 Protein Expression Analysis
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A549 cells seeded in 6-well plates at 1 × 106/well were infected with 1 mL of ΔS-VRP(G), and at 18 hpi, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer and protein samples were separated on a 4 to 15% gradient SDS-PAGE gel (Bio-Rad). As controls, A549-hACE2 cells were infected with SARS-COV-2 (Wuhan-Hu-1/2019) at a multiplicity of infection (MOI) of 3 and lysed in RIPA buffer. Western blot analysis was performed following the transfer of proteins onto a nitrocellulose membrane using mouse anti-nucleoprotein or S antibody (1:1,000; Sino Biological) and goat anti-mouse secondary antibody conjugated to horseradish peroxidase (GENA931; Sigma).
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