HEK293-TLR4 cells were treated with or without LPS (100 ng/ml) for 45–60 min. Cytosol, mitochondria, and nucleus fractions were isolated using a Cell Fractionation Kit (ab109719; Abcam) according to the manufacturer's protocol. ECSIT localization was determined by immunoblotting analysis with anti-ECSIT antibody (Abcam). THP-1 cells were transiently transfected with Flag-tagged ECSIT wt or Flag-tagged K372A expression vector. At 36 h posttransfection, the cells were treated or not with LPS (100 ng/ml) for different times. The cytosol and nucleus fractions were isolated using a Cell Fractionation Kit (Abcam). p65, p50, and Flag-ECSIT wt or Flag-ECSIT K372A localization was detected by Western blotting with anti-p65 (Cell Signaling Technology), anti-p50 (Cell Signaling Technology), and anti-Flag (Abcam) antibodies.
Cell fractionation kit
Manufactured by Abcam
Sourced in United States, United Kingdom
The Cell Fractionation Kit is a laboratory tool designed to separate and isolate different cellular components, such as nuclei, mitochondria, and other organelles, from a cell sample. It provides a standardized and efficient method for the fractionation of cellular contents.
Automatically generated - may contain errors
Lab products found in correlation
Protein assay rapid kit, by Fujifilm (7 mentions)
Ripa buffer, by Thermo Fisher Scientific (7 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Radioimmunoprecipitation assay buffer, by Thermo Fisher Scientific (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Nitrocellulose blotting membrane, by GE Healthcare (2 mentions)
Protease inhibitor, by Thermo Fisher Scientific (2 mentions)
G box gel imaging system, by Syngene (2 mentions)
Qubit 1x dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Qubit 4, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti p65, by Cell Signaling Technology (1 mentions)
Anti flag, by Abcam (1 mentions)
Anti p50, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Qproteome mitochondria isolation kit, by Qiagen (1 mentions)
Hepes, by Merck Group (1 mentions)
Odyssey clx infrared imaging system, by LI COR (1 mentions)
Ne per kit, by Thermo Fisher Scientific (1 mentions)
Orange mitochondrial membrane potential assay kit, by Abcam (1 mentions)
81 protocols using cell fractionation kit
1
Subcellular Localization of ECSIT
2
Quantification of Cobalt Internalization
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Internalization of the complexes in
different cellular factions (cytosolic, mitochondrial, or nuclear)
of A2780 cells was evaluated by using a cell fractionation kit (Abcam).
Cells were seeded in a 24-well plate at a density of 2 × 105 cells/mL and incubated for 24 h at 37 °C with 5% (v/v)
CO2. After the incubation, the culture media was replaced
by fresh medium with 10× the IC50 of the different
complexes or 0.1% (v/v) of DMSO, as for ICP-AES. Cells were then incubated
for 12 h at the same conditions as before. The culture media was recovered
to a new tube (supernatant fraction) and cells were detached from
the T-flask with 250 μL of TE (Gibco by life technologies).
Then, we followed the instructions provided by the manufacturer of
the cell fractionation kit (Abcam). At the end of the procedure, fresh
aqua regia was added to the supernatant tube and to the cellular fractions
and incubated overnight at room temperature (RT) in the hood fume.
Samples were delivered to Laboratório de Análises/LAQV
and the levels of cobalt were evaluated by ICP-AES.
different cellular factions (cytosolic, mitochondrial, or nuclear)
of A2780 cells was evaluated by using a cell fractionation kit (Abcam).
Cells were seeded in a 24-well plate at a density of 2 × 105 cells/mL and incubated for 24 h at 37 °C with 5% (v/v)
CO2. After the incubation, the culture media was replaced
by fresh medium with 10× the IC50 of the different
complexes or 0.1% (v/v) of DMSO, as for ICP-AES. Cells were then incubated
for 12 h at the same conditions as before. The culture media was recovered
to a new tube (supernatant fraction) and cells were detached from
the T-flask with 250 μL of TE (Gibco by life technologies).
Then, we followed the instructions provided by the manufacturer of
the cell fractionation kit (Abcam). At the end of the procedure, fresh
aqua regia was added to the supernatant tube and to the cellular fractions
and incubated overnight at room temperature (RT) in the hood fume.
Samples were delivered to Laboratório de Análises/LAQV
and the levels of cobalt were evaluated by ICP-AES.
3
Protein Extraction and Western Blot
4
Whole-cell lysate preparation protocol
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For preparing whole-cell lysate, SN12C and SN12L1 cells were washed twice with cold phosphate-buffered saline (PBS), harvested, and lysed with 0.1% sodium dodecyl sulfate (SDS)-added radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Tokyo, Japan) [49 (link)]. Cell fractions were extracted using a Cell Fractionation Kit (Abcam), according to the manufacturer’s instructions [50 (link)]. Protein assay was performed using a Protein Assay Rapid Kit (Wako Pure Chemical Corporation, Osaka, Japan).
5
Cytoplasmic Circ_0038467 Expression Analysis
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Nucleus and cytoplasm fractions were prepared using a Cell Fractionation Kit purchased from Abcam (ab109719). All operations were performed following the manufacturer’s instructions. Both fractions were used to isolate RNA samples, which were subjected to RT-PCRs to determine the expression of Circ_0038467. GAPDH was used as a cytoplasm marker.
6
Cell Fractionation for Protein Analysis
7
Whole Cell Lysate Preparation and Fractionation
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For preparing whole cell lysate, HSC3 and HSC4 cells were washed twice with cold PBS, harvested and lysed with 0.1% SDS-added RIPA-buffer (Thermo Fisher Scientific, Tokyo, Japan) [32 (link)]. Cell fractions were extracted by using a Cell Fractionation Kit (Abcam, Cambridge, MA, USA), according to the manufacturer’s instructions [36 (link)]. Protein assay was performed using a Protein Assay Rapid Kit (Wako Pure Chemical Corporation, Osaka, Japan).
8
Cellular Fractionation of THP-1 Cells
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Cellular fractionation assay was performed as previously described (19 (link), 24 (link)). Briefly, THP-1 cells were treated with or without LPS (200 ng/ml) for different times. Cytosol and mitochondria fractions were isolated using a Cell Fractionation Kit (Ca# ab109719, Abcam), according to the manufacturer's protocol. Immunoblotting analysis was performed with anti-TRAF6, anti-CRBN, anti-IκB-α, and anti-GRIM19 antibodies.
9
Subcellular Fractionation and Immunoblot
10
Subcellular Protein Fractionation and Analysis
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Cytoplasmic and nuclear lysates were prepared using the Cell Fractionation Kit (Abcam, Cambridge, MA). Whole cell lysates and OS xenograft tumor lysates were prepared in RIPA buffer (Invitrogen) containing protease and phosphatase inhibitors (ThermoFisher Scientific, Carlsbad, CA). Lysates were sonicated and cleared of cell debris by centrifugation. The supernatants were used to quantify total protein (Pierce protein assay). Equal amounts of protein were separated by SDS-PAGE, transferred to nitrocellulose membrane and subjected to immunoblot analysis with primary antibodies. Images were captured using the LI-COR (Lincoln, NE) and relative quantification was performed using Image J (NIH, Bethesda, MD).
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