Protein lysates were prepared by pelleting cells (3×106) and subjecting them to lysis using 200 μL of Mammalian Cell Lysis Buffer (G-Biosciences; St. Louis, MO) and 1 μL of protease and phosphatase inhibitor Cocktail (Roche Diagnostics; Basel, Switzerland). The lysates were subjected to polyacrylamide gel electrophoresis and then transferred to PVDF membrane. The membranes were then blocked with 5% milk and incubated with primary (listed in Supplementary Table 3 ) and fluorescent tagged secondary antibodies. After washes the membranes scanned using Odyssey fluorescent imager/scanner (LI-COR).
Mammalian cell lysis buffer
Manufactured by G Biosciences
Sourced in United States
Mammalian Cell Lysis Buffer is a solution designed to facilitate the disruption and lysis of mammalian cells. The buffer contains a combination of ionic and non-ionic detergents, as well as other components, that work together to solubilize cell membranes and release the contents of the cells, including proteins, nucleic acids, and other biomolecules.
Automatically generated - may contain errors
3 protocols using mammalian cell lysis buffer
1
Western Blot Protocol for Protein Detection
2
Protein Expression Analysis Protocol
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The sample was homogenized and lysed in cell lysis buffer (Mammalian cell lysis buffer, Gbiosciences cat no. 786-180, U.S.A) on ice and centrifuged at 12000 × g for 30 min at 4 °C. Protein estimation was carried out through the Bicinchoninic Acid method (BCA) (Gbiosciences cat no. 786846, U.S.A). An equal amount of protein samples were loaded on SDS-PAGE and transferred to the nitrocellulose membrane. 5% skim milk was used for blocking after which it was probed with primary antibodies. Subsequently, the membrane was washed and labeled with a horseradish peroxidase-conjugated secondary antibody. The image was captured using enhanced chemiluminescence western blotting substrate (Millipore cat no. WBKLS0100, U.S.A) and detected with ImageQuant LAS 4000 (GE Healthcare Life sciences). The list of antibodies is mentioned in Table S2 .
3
Western Blot Analysis of Adipose Tissue
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As described previously (58 (link)), adipose tissue was homogenized using bead beater in mammalian cell lysis buffer (G Biosciences) supplemented with protease and phosphatase inhibitors. Samples were centrifuged twice to isolate the clear protein fraction. An equal amount of protein (μg) was used to prepare SDS-PAGE samples. After adding the loading dye, samples were resolved on SDS-PAGE and subsequently transferred to the polyvinylidene difluoride membrane. The membrane was then blocked and probed with primary overnight followed by washing three times with TBS with Tween-20 buffer and secondary antibodies (diluted in 4% BSA in TBS with Tween-20) for the next 2 hrs. Finally, the blots were developed using HRP substrate (Immobilon Western Chemiluminescent HRP substrate, Millipore) and visualized under chemiluminescence, and images were captured using BioRad Imager.
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