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Mammalian lysis buffer

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Mammalian lysis buffer is a reagent used to extract and solubilize proteins from mammalian cells. It contains a mixture of detergents, buffers, and other components that facilitate the disruption of cell membranes and release of intracellular proteins.

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Lab products found in correlation

Firefly luciferase, by Promega (2 mentions) Luciferin, by Promega (2 mentions) Genjet reagent, by SignaGen (1 mentions) Glomax luminometer, by Promega (1 mentions) Glowmax luminometer, by Promega (1 mentions) Genjet, by SignaGen (1 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)

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Mammalian cell lysis buffer (3 citations)

3 protocols using mammalian lysis buffer

1

Quantifying Protein-Mediated Luciferase Activity

Cited 1 time
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Luciferase assays were performed as described.9 (link), 19 (link) Briefly, 24 well plates were seeded with 5 × 104 Hela PKRKD cells per well 16 hours prior to the experiment. Cells were transfected with 200 ng of the indicated PKR plasmid, the indicated amount of K3 and E3 orthologs, and 50 ng of firefly luciferase (Promega) using GenJet reagent (Signagen) at a DNA to GenJet ratio of 1:2. Empty pSG5 vector was used to maintain the DNA concentration where appropriate. At 48 hours post-transfection, cells were lysed with mammalian lysis buffer (Goldbio) and luciferin (Promega) was added following the manufacturer’s recommendations. Luciferase activity was measured using a GloMax luminometer (Promega). Experiments were conducted in triplicate for each of three independent experiments.
Park C., Peng C., Brennan G, & Rothenburg S. (2019). Species-specific inhibition of antiviral protein kinase R by capripoxviruses and vaccinia virus. Annals of the New York Academy of Sciences, 1438(1), 18-29.
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2

Luciferase Assay for PKR Plasmid Activity

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Luciferase assays were performed following the protocol described previously [45 (link)]. Briefly, HeLa-PKRkd cells (5 × 104 cells/well) were seeded in 24-well plates a day before the transfection. For each transfection reaction mix, 0.05 µg of firefly luciferase (Promega) and 0.2 µg of each of the indicated PKR plasmids were co-transfected with 0.8 µg of either E3L, CRV157, CRV157-K48A/K49A, CRV157 del C, or CRV157 del N plasmids using GenJet (SignaGen Laboratories, Frederick, MD), with transfection reagent to DNA ratios of 2:1. For the titration experiments, the indicated concentrations of CRV157 or CRV157 del C plasmids were co-transfected. Each transfection was performed in triplicate, and plasmid DNA concentrations for each transfection mix were kept constant by adding an appropriate amount of pSG5 vector plasmid where necessary. After 48 h, whole-cell lysates were extracted using mammalian lysis buffer (GoldBio, St Louis, MO), USA, followed by the addition of luciferin (Promega, Madison, WI, USA). Luciferase activity was determined in a Glowmax luminometer (Promega). Each experiment was conducted at least three times, and representative experiments are shown.
Rahman M.J., Tazi L., Haller S.L, & Rothenburg S. (2022). Crocodilepox Virus Protein 157 Is an Independently Evolved Inhibitor of Protein Kinase R. Viruses, 14(7), 1564.
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3

ARPE-19 Cell Transfection Protocol

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ARPE-19 cells were seeded in six-well plates at a density of 0.5 × 106 cells/well in antibiotic-free normal growth cell culture medium to achieve 70% to 80% confluence on day 2. For transfection of each well 12 μl of transfection reagent (Lipofectamine RNAiMax, Invitrogen) was diluted in 200 μl of in serum-free basal medium, vortexed and incubated for 45 min. Then of 12 μL siRNA duplex (from 10 μM stock either scrambled or p53) were added and incubation continued for another 30 to 45 min at room temperature. The cell monolayer was rinsed in serum free-basal cell culture medium, and the siRNA-transfection mixture was added dropwise onto the monolayer and incubated for 24 hours at 37 °C. Fresh complete cell culture medium containing 10% FBS heat-inactivated serum was added onto the monolayer without removing the transfection mixture followed by incubation for an additional 24 h. Total cell lysates for Western blot analysis were prepared using GoldBio Mammalian Lysis Buffer (St. Louis, MO) instead of RIPA lysis buffer. Protein concentrations were determined by the BCA protein quantitation assay.
Linetsky M., Guo J., Udeigwe E., Ma D., Chamberlain A.S., Yu A.O., Solovyova K., Edgar E, & Salomon R.G. (2020). 4-Hydroxy-7-oxo-5-heptenoic acid (HOHA) lactone induces apoptosis in retinal pigment epithelial cells. Free radical biology & medicine, 152, 280-294.
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