Subcellular fraction was obtained from cultured cells and tissues using Subcellular Protein Fractionation Kit for Cultured Cells and Subcellular Protein Fractionation Kit for Tissues (Thermo Fisher Scientific).
Subcellular protein fractionation kit for tissue
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Subcellular Protein Fractionation Kit for Tissues is a laboratory equipment designed to separate and extract proteins from different organelles and cellular compartments of tissue samples. The kit provides a systematic process to isolate and purify proteins from the nucleus, cytoplasm, mitochondria, and other subcellular fractions.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Subcellular protein fractionation kit for cultured cells, by Thermo Fisher Scientific (2 mentions)
Horse serum, by Thermo Fisher Scientific (2 mentions)
Mycoprobe mycoplasma detection kit, by R&D Systems (2 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (2 mentions)
Amersham ecl prime, by GE Healthcare (2 mentions)
Imagequant las 4000 mini, by GE Healthcare (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Imagequant las 4000 system, by GE Healthcare (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Quick start bradford dye reagent, by Bio-Rad (1 mentions)
Ab 302613, by Abcam (1 mentions)
Las 3000, by Fujifilm (1 mentions)
Ab5095, by Abcam (1 mentions)
Ab5408, by Abcam (1 mentions)
Ab817, by Abcam (1 mentions)
33 protocols using subcellular protein fractionation kit for tissue
1
Subcellular Protein Fractionation
2
Subcellular Protein Fractionation and Validation
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Subcellular fractions from cortices of 3 months old mice were generated by using the Subcellular Protein Fractionation Kit for Tissues (Thermo Fisher Scientific) according to the manufacturer’s recommendations. Protein concentrations were determined via Bradford Assay using the QuickStart™ Bradford Dye Reagent (Bio-Rad, Hercules, CA, USA). To validate purity, 10 μg of each subcellular fraction were processed by a standard SDS-PAGE-based western blot protocol. After blocking of non-specific epitopes, the membrane was incubated with primary antibodies against GAPDH (1:8,000; RRID:AB_561053; Cell Signaling Technology, Danvers, MA, USA) and Histone H3 (1:10,000; RRID:AB_302613; Abcam, Cambridge, ENG, UK) overnight at 4°C. Afterwards, the membrane was washed in TBS supplemented with 0.1% Tween®20 prior to incubation with the appropriate HRP-conjugated secondary antibody (1:5,000; RRID:AB_631746; Santa Cruz Biotechnology, Dallas, TX, USA). Following washing, the bands were detected by chemiluminescence using Immobilon Western HRP Substrate solutions (Merck) and documented by the LAS3000 (Fujifilm, Düsseldorf, Germany) and the corresponding software.
3
C2C12 Cell Differentiation and Fractionation
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C2C12 cells (ATCC CRL-1772) were cultured in Dulbecco's modified Eagle medium (D-MEM; Thermo Fisher) supplemented with 20% fetal bovine serum (HyClone, GE Healthcare Life Systems) and 1× penicillin/streptomycin (Thermo Fisher). Cells were tested for mycoplasma by using a Myco Probe mycoplasma detection kit (R&D Systems). To induce differentiation, confluent cells were treated with differentiation media containing D-MEM supplemented with 2% horse serum (Thermo Fisher) and 1× penicillin/streptomycin.
For immunoprecipitation studies, cells were differentiated for 7 days. For Dmd knockdown studies, Lipofectamine RNAiMax (Thermo Fisher) was used to transfect siRNA into differentiated cells 2 days before harvest. Yap5SA (pCMV-Flag Yap2) or GFP plasmid was transfected into C2C12 cells by using Lipofectamine 3000 (Thermo Fisher) one day before the differentiation treatment was started. For subcellular fractionation, cells were differentiated for 3 days and treated with siRNA at day 1 of differentiation for 48 hours before harvest. Nuclear, cytoplasmic, and plasma membrane fractions of cellular extracts were prepared by using a subcellular protein fractionation kit for tissues (Thermo Fisher) according to the manufacturer's instructions.
For immunoprecipitation studies, cells were differentiated for 7 days. For Dmd knockdown studies, Lipofectamine RNAiMax (Thermo Fisher) was used to transfect siRNA into differentiated cells 2 days before harvest. Yap5SA (pCMV-Flag Yap2) or GFP plasmid was transfected into C2C12 cells by using Lipofectamine 3000 (Thermo Fisher) one day before the differentiation treatment was started. For subcellular fractionation, cells were differentiated for 3 days and treated with siRNA at day 1 of differentiation for 48 hours before harvest. Nuclear, cytoplasmic, and plasma membrane fractions of cellular extracts were prepared by using a subcellular protein fractionation kit for tissues (Thermo Fisher) according to the manufacturer's instructions.
4
Nuclear Fractionation of Hippocampus
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Nuclear fractionation from hippocampus of 30‐week‐old wild‐type and R6/1 mice was performed using a Subcellular Protein Fractionation Kit for Tissues (Thermo Fisher Scientific, #87790) following manufacturer’s instructions. Chromatin‐bound and nuclear soluble fractions were obtained and examined by Western blot as described above.
5
C2C12 Cell Differentiation and Fractionation
6
Protein Immunoprecipitation and Membrane Fractionation
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The tissues were lysed in radioimmune precipitation buffer (RIPA) with protease and phosphatase inhibitor cocktails (Roche Applied Sciences) and 1 mM sodium orthovanadate. After quantification of total protein concentration, equal amounts of protein (amount in 150 μg) were precipitated with the PDI, PP1, PP2A or PICK1 antibody and subsequently incubated with protein G sepharose at 4 °C overnight. Beads were collected, eluted in sample buffer and boiled at 95 °C for 5 min. The elution step was performed under the same conditions and in parallel. To analyze membrane expressions of GluA1 and GluA2, subcellular Protein Fractionation Kit for Tissues (Thermo Scientific, USA) was used according to the manufacturer’s instructions. Next, Western blot was performed according to standard procedures6 (link),10 (link),18 (link),19 (link),21 (link).
7
Subcellular Protein Fractionation of Liver Tissue
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Liver tissue samples were homogenized and fractionated to cytosolic and nuclear fractions using a Subcellular Protein Fractionation Kit for Tissues (Thermo Fisher Scientific, Waltham, MA, USA). Samples from 6 mice/group were used in the experiments. Briefly, the stored frozen tissues were thawed and homogenized in protease inhibitor-containing buffers. The cytoplasmic and nuclear fractions were separately collected by centrifugation according to the manufacturer’s instructions. The concentration of the cytoplasmic and nuclear protein fractions obtained were estimated and stored at −70 °C.
8
Subcellular Fractionation of Murine Livers
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Control and LKO livers of mice 3- and 8-mo post-DEN exposure were harvested. Cytoplasmic and nuclear fractions were prepared from these livers using the Thermo Scientific Subcellular Protein Fractionation Kit for Tissues (Cat. No. 87790), according to the manufacturer’s recommendations.
9
Subcellular Protein Fractionation and Western Blot Analysis
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Cytoplasmic, soluble nuclear, and chromatin‐bound protein extracts were prepared by stepwise separation using Subcellular Protein Fractionation Kit for Tissues (Thermo Fisher Scientific, 87790). For this purpose, whole livers were harvested, snap‐frozen in liquid nitrogen, and stored at −80°C. Liver tissue was homogenized in liquid nitrogen using mortar and pestle and by avoiding thawing of the tissue at any time. 200 mg of each homogenized liver sample was resuspended in 2 ml of ice‐cold Cytoplasmic Extraction Buffer (CEB) supplemented with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, 87786 at 1:100) and 1 mM sodium metavanadate (NaVO3) and processed as outlined by the manufacturer's protocol. Fractions were run on western blots and incubated with antibodies against Pol II (Abcam, ab817), Pol II Ser2 phospho (Abcam, ab5095), and Pol II Ser 5 phospho (Abcam, ab5408).
10
Subcellular Protein Fractionation and Analysis
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Cell fractions of 50 (100 mg) stage 17 wild-type or injected embryos were obtained using the Subcellular Protein Fractionation Kit for Tissues (Thermo Fisher Scientific # 87790). Protein quantification was performed using Bradford technique (Pierce). Around 40 μg of proteins were loaded per lane of a 4% to 12% precast gel (Bio-Rad Mini-PROTEAN). Proteins were transferred on PVDF-membrane and blocked for 1 h with 5% skimmed milk. The following antibodies were used: anti-GFP 1:1,000 (Torrey Pines BioLabs; TP-401), anti-Tubulin 1:1,000 (Sigma, T9026), anti-rabbit-HRP, or anti-mouse-HRP 1:10,000 (Millipore).
The numerical data used in all figures are included inS1 Data .
The numerical data used in all figures are included in
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