Cell fractionation kit

Manufactured by Cell Signaling Technology

Sourced in United States

The Cell Fractionation Kit is a laboratory tool designed to separate and isolate different cellular components, such as organelles, proteins, and other macromolecules, from a homogenized cell sample. The kit provides the necessary reagents and protocols to perform differential centrifugation, a widely used technique for cellular fractionation.

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Lab products found in correlation

Ripa buffer, by Cell Signaling Technology (16 mentions) Anti α tubulin antibody, by Merck Group (9 mentions) Nupage bis tris gel, by Thermo Fisher Scientific (6 mentions) Anti α tubulin antibody, by Cell Signaling Technology (5 mentions) Pvdf membrane, by Merck Group (4 mentions) Subcellular protein fractionation kit, by Thermo Fisher Scientific (4 mentions) Bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Anti histone h3 rabbit polyclonal ab, by Merck Group (3 mentions) Anti nf κb p65 rabbit mab d14e12, by Cell Signaling Technology (3 mentions) Anti histone h3, by Cell Signaling Technology (3 mentions) P mst1 2, by Cell Signaling Technology (3 mentions) P lats1, by Cell Signaling Technology (3 mentions) Protease inhibitor cocktail, by Euroclone (3 mentions) Mini trans blot turbo rta system, by Bio-Rad (3 mentions) Mini protean tetra cell system, by Bio-Rad (3 mentions) Quantity one software, by Bio-Rad (3 mentions) Polyvinylidene difluoride membrane, by Merck Group (2 mentions) Goat anti rabbit igg hrp linked ab, by Cell Signaling Technology (2 mentions) Bcl xl, by Abcam (2 mentions) Odyssey infrared imaging system, by LI COR (2 mentions)

112 protocols using cell fractionation kit

Quantitative Western Blot Analysis of MEFs

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MEFs were grown to 80–90% confluency then collected. Cell pellets were weighed and then resuspended in a proportional volume of phosphate buffered saline (PBS). Equivalent volumes of resuspension were always used for each lysis. Cells were lysed using a cell fractionation kit (Cell Signaling Technologies, #9038) with each buffer supplemented with 1 mM PMSF and 1x protease inhibitor cocktail (Cell Signaling Technologies, #5871).
Lysates were separated by SDS-PAGE on 3–8% Tris-acetate gels (BioRad #3450129) for 2.5 hr at 150V, then transferred to PVDF membrane by wet transfer at 50V for 1 hr. Membranes were blocked in either 5% milk or 5% BSA according to the recommendations of the primary antibody manufacturer. Primary antibodies were as follows: anti-NKCC1 (Cell Signaling Technologies #14581), anti-TCF11/NRF1 (Cell Signaling Technologies #8052). Membranes were incubated in primary antibody at 1:1000 in blocking buffer overnight. IRDye secondary antibody (Abcam #216773) was used for infrared detection at 1:10,000 dilution in blocking buffer for 1 hr. Membranes were scanned on an Odyssey CLx (Li-cor) and analyzed with the accompanying software, Image Studio.

Talsness D.M., Owings K.G., Coelho E., Mercenne G., Pleinis J.M., Partha R., Hope K.A., Zuberi A.R., Clark N.L., Lutz C.M., Rodan A.R, & Chow C.Y. (2020). A Drosophila screen identifies NKCC1 as a modifier of NGLY1 deficiency. eLife, 9, e57831.

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Cell Fractionation Protocol

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Cells were harvested using EDTA and then counted to adjust cell numbers for each sample. Fractionation was performed using Cell fractionation kit (#9038, Cell Signaling Technology) or Subcellular Protein Fractionation Kit (#78840, Thermo Fisher Scientific). The pellet was washed one time using cold PBS between every step.

Gao Y., Nihira N.T., Bu X., Chu C., Zhang J., Kolodziejczyk A., Fan Y., Chan N.T., Ma L., Liu J., Wang D., Dai X., Liu H., Ono M., Nakanishi A., Inuzuka H., North B.J., Huang Y.H., Sharma S., Geng Y., Xu W., Liu X.S., Li L., Miki Y., Sicinski P., Freeman G.J, & Wei W. (2020). Acetylation-dependent regulation of PD-L1 nuclear translocation dictates the efficacy of anti-PD-1 immunotherapy. Nature cell biology, 22(9), 1064-1075.

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Endosomal Trafficking Lipid Composition

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DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), bovine liver phosphatidylinositol (PI), brain PS, and egg PA were purchased from Avanti Polar Lipids. PI 3-phosphate diC16 [PI(3)P], PI 4-phosphate diC16 [PI(4)P], and PI 5-phosphate diC16 [PI(5)P] were obtained from Echelon Biosciences. Folch fraction I and egg PC were purchased from Sigma-Aldrich. YM201636 PIKfyve inhibitor was purchased from Cayman Chemical. A cell fractionation kit was obtained from Cell Signaling Technology. Nontargeting control siRNA was obtained from Horizon Discovery, and human KLC1/2 siRNAs were from Santa Cruz Biotechnology. The following primary antibodies were used: horseradish peroxidase (HRP)–conjugated anti-His⁶ (71841, Novagen), anti-GFP for immunoblotting (3E1, Roche), anti-actin (AC-74, Sigma-Aldrich), anti–β-tubulin (AA2, Sigma-Aldrich), anti–histone H3 (Abcam), anti-giantin (PRB-114C, Covance), anti-HA (HA-7, Sigma-Aldrich), anti-KLC1 ([EPR12441(B)], Abcam), anti-KLC2 (Abcam), anti-KIF5B (Abcam), anti-LAMP1 (lysosome-associated membrane protein 1) (D2D11, Cell Signaling Technology), anti-Rab7 (D95F2, Cell Signaling Technology), and anti-Rab6 (D37C7, Cell Signaling Technology). Alexa 568– and Alexa 633–conjugated anti-mouse or anti-rabbit secondary antibodies were from Thermo Fisher Scientific.

Antón Z., Weijman J.F., Williams C., Moody E.R., Mantell J., Yip Y.Y., Cross J.A., Williams T.A., Steiner R.A., Crump M.P., Woolfson D.N, & Dodding M.P. (2021). Molecular mechanism for kinesin-1 direct membrane recognition. Science Advances, 7(31), eabg6636.

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Subcellular Fractionation of Proteins

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Protein subcellular fractionation was performed using the Cell Fractionation kit according to the manufacturer’s instructions (Cell Signaling Technology, Danvers, MA, USA). Briefly, cells were washed with cold 1× PBS and scratched to harvest the pellets. The pellets were lysed in 500 uL cytoplasmic isolation buffer (CIB) and incubated on ice for 5 min. The lysate was centrifuged for 5 min at 500× g at 4 °C. The supernatant was the cytoplasmic fraction. The pellets were resuspended with 500 uL membrane isolation buffer (MIB) and incubated on ice for 5 min. The lysate was centrifuged for 5 min at 500× g at 4 °C. The supernatant was the membrane fraction. The pellets were resuspended with cytoskeleton/nucleus isolation buffer and sonicated for 5 s three times. The resulting fraction was the cytoskeleton/nuclear fraction. Three subcellular fractions were resolved in 10% SDS-PAGE followed by Western blotting analysis.

Chu P.Y., Tung S.L., Tsai K.W., Shen F.P, & Chan S.H. (2020). Identification of the Novel Oncogenic Role of SAAL1 and Its Therapeutic Potential in Hepatocellular Carcinoma. Cancers, 12(7), 1843.

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Isolation and Fractionation of Mitochondria and Nuclei

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Intact mitochondria were isolated from cells and tissues using a Mitochondria Isolation Kit for Cultured Cells and a Mitochondria Isolation Kit for Tissue (both Thermo Fisher), respectively, according to the manufacturer's protocols. Mitochondrial proteins were extracted by lysis of mitochondria in mammalian protein extraction reagent (Thermo Fisher). Nuclei were isolated using a Nuclei Isolation Kit (Biohao, Shanghai, China) according to the manufacturer's instructions. To detect subcellular distribution of AIF, cells were separated into three distinct fractions: cytoplasmic, organelle (mitochondria), and nuclear/cytoskeletal, using a Cell Fractionation Kit (#9038, Cell Signaling Technology) according to the manufacturer's instructions.

Li H.S., Zhou Y.N., Li L., Li S.F., Long D., Chen X.L., Zhang J.B., Feng L, & Li Y.P. (2019). HIF-1α protects against oxidative stress by directly targeting mitochondria. Redox Biology, 25, 101109.

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Cell Fractionation for Cytosolic and Membrane Proteins

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Subcellular fractions, such as cytosolic and membrane, were prepared using the Cell Fractionation Kit (Cell Signaling Technology) following the manufacturer's instructions. The methodology is based on differential centrifugation as described by DerMardirossian et al. [27 (link)].

Prasad R., Kappes J.C, & Katiyar S.K. (2016). Inhibition of NADPH oxidase 1 activity and blocking the binding of cytosolic and membrane-bound proteins by honokiol inhibit migratory potential of melanoma cells. Oncotarget, 7(7), 7899-7912.

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Quantifying HIF-1α Protein Activity

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To compare the activity of HIF-1α WT proteins between mutants A and B and the control under normoxic conditions (20% O₂), the following five groups were transfected into NP cells using Lipofectamine (Invitrogen, Waltham, MA, USA): (1) Desferrioxamine (DFO) only, (2) pcDNA3 only, (3) pHIF-1α WT, (4) pHIF-1α Mut A, and (5) pHIF-1α Mut B. DFO was used as a positive control, because it inhibits both PHD enzyme and FIH-1 enzyme activities [41 (link)]. The transfected cells were lysed using a cell fractionation kit (Cell Signaling Technology, Danvers, MA, USA). The cells were separated into nuclear and cytoplasmic fractions in Nupage 4–12% Bis-Tris gels (Invitrogen, Waltham, MA, USA) and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk and washed with phosphate-buffered saline and 0.2% Tween 20 buffer. The membranes were blotted with mouse anti-HIF-1α (Cell Signaling Technology, Danvers, MA, USA), mouse anti-nuclear lamin A/C, or anti-α-tubulin (Santa Cruz Biotechnology, Dallas, TX, USA) antibodies. The blots were treated with goat anti-mouse conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA) and visualized using an enhanced chemiluminescence system (Uppsala, Sweden).

Kim J.W., An H.J., Yeo H., Jeong Y., Lee H., Lee J., Nam K., Lee J., Shin D.E, & Lee S. (2021). Activation of Hypoxia-Inducible Factor-1α Signaling Pathway Has the Protective Effect of Intervertebral Disc Degeneration. International Journal of Molecular Sciences, 22(21), 11355.

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Protein Fractionation and Immunoblotting

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Nuclear/cytoplasmic fractionation was separated using Cell Fractionation kit according to the manufacturer's instructions, and the whole cell lysates were extracted using RIPA buffer (both from Cell Signaling Technology, Beverly, MA, USA). Western blotting was performed according to a standard method, as previously described (28 (link)). Antibodies against YAP, TAZ, Bcl-2 and Bcl-xL were purchased from Abcam (Cambridge, MA, USA). The membranes were stripped and reprobed with an anti-α-tubulin antibody (Sigma-Aldrich, St. Louis, MO, USA) as the loading control.

Que K., Tong Y., Que G., Li L., Lin H., Huang S., Wang R, & Tang L. (2017). Downregulation of miR-874-3p promotes chemotherapeutic resistance in colorectal cancer via inactivation of the Hippo signaling pathway. Oncology Reports, 38(6), 3376-3386.

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Effective Cell Fractionation for Protein Isolation

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The Cell Fractionation Kit (no. 9038, Cell Signaling Technology) was used for cell fractionation according to the manufacturer’s protocol and the published works (23 (link), 24 (link)). Briefly, cells were collected by scraping, washed in PBS, and pelleted (350g, 5 min). The counted cells were resuspended in 500 μl of PBS and 100 μl was reserved for whole-cell lysis in low-salt lysis buffer, and the remaining cell pellet was subjected to cell fractionation. Cytoplasmic proteins were isolated using Cytoplasm Isolation Buffer (CIB) buffer, and integral membrane and organellular membrane proteins were isolated using Membrane Isolation Buffer (MIB). Cytosol-located TUBULIN and cytomembrane-located Na⁺K⁺-ATPase were used to demonstrate the efficiency of the cellular fractionation.

Hu Z., Xie Y., Lu J., Yang J., Zhang J., Jiang H., Li H., Zhang Y., Wu D., Zeng K., Bai X, & Yu X. (2023). VANGL2 inhibits antiviral IFN-I signaling by targeting TBK1 for autophagic degradation. Science Advances, 9(25), eadg2339.

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Cell Fractionation and Real-time PCR

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Cytoplasmic and nuclear fractions were prepared using the Cell Fractionation kit (Cell Signaling Technology, Danvers, MA, USA; Cat. #9038), according to the manufacturer’s protocol. Real-time PCR analysis was performed to determine cytoplasmic marker ACTB and nuclear markers U6.

Li H., Zhan J., Zhao Y., Fan J., Yuan S., Yin Z., Dai B., Chen C, & Wang D.W. (2019). Identification of ncRNA-Mediated Functions of Nucleus-Localized miR-320 in Cardiomyocytes. Molecular Therapy. Nucleic Acids, 19, 132-143.

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