PARTITION & CONCENTRATE nuclear and cytoplasmic proteins in 45 min for EMSA, Western blot & translocation assays with cells or tissues. For 300+ isolations based on a well in a 6-well dish, or 100 isolations with 10 cm dishes. This nuclear protein extraction kit partitions and concentrates proteins in nuclei and cytosol in a simple 45 minute protocol using a microcentrifuge.
Simplicity: Allows for simultaneous isolations of multiple samples.
Scalability: Suitable for 300+ isolations/extractions based on a well in a 6-well dish, or 100 isolations/extractions using semi-confluent 10 cm dishes.
Versibility: Applicalbe for cells from virtually any mammalian species, or with tissues using an initial homogenization step.
Broad Downstream Applicability: Ideal for nuclei isolation, nuclei-cytosol fractionation, nuclear protein extraction, measurement of nuclear import, EMSA, transcription factor activation and translocation assays.
Nuclear Protein Isolation (Fractionation-Translocation) Kit: Overview
Application to transcription factor import and isolation, and nuclear protein isolation
A key regulatory event in transcription factor activation is translocation from cytosol to nucleus. Proteins with exposed targeting signals interact with the nuclear pore import complex and enter into the nucleus to perform regulatory functions. The Nuclear Protein Isolation Kit provides a convenient method to isolate nuclear and cytoplasmic proteins, and detect and quantitate cytoplasmic to nuclear import.
Downstream applications for the nuclear protein isolation kit include, but are not limited to, quantitation of transcription factor activation, EMSA, cellular localization studies, nuclear protein isolation, and general investigative mechanisms of nuclear import.
Application to peptide fragment import
Transmembrane proteins, and other polypeptides, with nuclear import signals may become proteolytically cleaved and translocate through the nuclear pore to bind chromosomal regulatory elements. The nuclear protein isolation protocol provided with this kit can also be applied to identify and quantitate the nuclear import of the generated peptide fragments.
Protocol Synopsis: Nuclear Protein Translocation-Isolation
To separate cytoplasmic from nuclear fraction, cells are first exposed to the "Cytoplasmic Isolation Reagent." The cell suspension is briefly centrifuged in a microcentrifuge, which generates the cytoplasmic fraction in the supernatant. The resulting pellet is washed with the Cytoplasmic Isolation Reagent, which generates isolated nuclei. The nuclear proteins are then extracted with the "Nuclear Isolation Reagent.” Aliquots of each fraction can be resolved in Western blots, with consecutive lanes loaded to display total cell lysate, cytoplasmic and nuclear fraction.
Figure Legends
Examples of data derived from the Fivephoton Biochemicals Nuclear Protein Isolation Kit. Fig. 1 demonstrates fractionation of the cytosolic protein GAPDH and the nuclear protein p84. View Images and Data Tab for data enlargements.
Fig 2. reveals nuclear and cytoplasmic fractionation and translocation of the ER stress-related transcription factor ATF6 into the nucleus. Expression of a mutant protein (Mu) creates cellular stress and results in ATF6 cleavage and translocation of an active-cleaved transcription factor fragment (CL) from the cytoplasm (CYT) into the nucleus (Nuc). Expression of the WT protein maintains full length uncleaved (FL) ATF6 in the cytoplasm.
Product Citations
Snodgrass SM, Cihil KM, Cornuet PK, Myerburg MM, Swiatecka-Urban A (2013). Tgf-β1 Inhibits Cftr Biogenesis and Prevents Functional Rescue of ΔF508-Cftr in Primary Differentiated Human Bronchial Epithelial Cells. http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0063167. Link to article
Ramirez-Sanchez I, et al. (2013). (-)-Epicatechin rich cocoa mediated modulation of oxidative stress regulators in skeletal muscle of heart failure and type 2 diabetes patients. Int J Cardiol. 2013 Oct 9;168(4):3982-90. doi: 10.1016/j.ijcard.2013.06.089. Epub 2013 Jul 17. Link to article
Lijia Ma et. al. 2014. Ginsenoside Rb3 Protects Cardiomyocytes against Ischemia-Reperfusion Injury via the Inhibition of JNK-Mediated NF-κB Pathway: A Mouse Cardiomyocyte Model. Plos One DOI: 10.1371/journal.pone.0112986. Link to article
Ryou, M. G., Choudhury, G. R., Li, W., Winters, A., Yuan, F., Liu, R., & Yang, S. H. (2015). Methylene blue-induced neuronal protective mechanism against hypoxia-reoxygenation stress. Neuroscience, 301, 193-203. Link to article
Chen, Y. H., Lin, W. W., Liu, C. S., Hsu, L. S., Lin, Y. M., & Su, S. L. (2016). Caveolin-1 Expression Ameliorates Nephrotic Damage in a Rabbit Model of Cholesterol-Induced Hypercholesterolemia. PLOS ONE, 11(4), e0154210: Link to artile
Hsieh, CL et al. 2017. Reactive oxygen species-mediated switching expression of MMP-3 in stromal fibroblasts and cancer cells during prostate cancer progression. SciRep. 2017 Aug 22;7(1):9065. doi: 10.1038/s41598-017-08835-9. Link to article
Sanders, K.A., Delker, D.A., Huecksteadt, T. et al. (2019). RAGE is a Critical Mediator of Pulmonary Oxidative Stress, Alveolar Macrophage Activation and Emphysema in Response to Cigarette Smoke. Sci Rep 9, 231: Link to article
Nano, E., Petropavlovskaia, M. & Rosenberg, L. Islet neogenesis associated protein (INGAP) protects pancreatic β cells from IL-1β and IFNγ-induced apoptosis. 2021. Cell Death Discov. 7, 56Link to article. https://doi.org/10.1038/s41420-021-00441-z
Protocol
Representative References on Nuclear Protein Translocation and Isolation
Al-Hanbali M, Ali D, Bustami M, Abdel-Malek S, Al-Hanbali R, et al. 2009. Epicatechin suppresses IL-6, IL-8 and enhances IL-10 production with NF-kappaB nuclear translocation in whole blood stimulated system. Neuro Endocrinol Lett 30: 131-8.
Allen PB, Kwon YG, Nairn AC, Greengard P. 1998. Isolation and characterization of PNUTS, a putative protein phosphatase 1 nuclear targeting subunit. J Biol Chem 273: 4089-95.
Anderson LG, Meeker RB, Poulton WE, Huang DY. 2009. Brain distribution of carboxy terminus of Hsc70-interacting protein (CHIP) and its nuclear translocation in cultured cortical neurons following heat stress or oxygen-glucose deprivation. Cell Stress Chaperones.
Arminan A, Gandia C, Bartual M, Garcia-Verdugo JM, Lledo E, et al. 2009. Cardiac differentiation is driven by NKX2.5 and GATA4 nuclear translocation in tissue-specific mesenchymal stem cells. Stem Cells Dev 18: 907-18.
Azuma Y, Tabb MM, Vu L, Nomura M. 1995. Isolation of a yeast protein kinase that is activated by the protein encoded by SRP1 (Srp1p) and phosphorylates Srp1p complexed with nuclear localization signal peptides. Proc Natl Acad Sci U S A 92: 5159-63.
Brown TL, Rosen JM. 1986. Isolation and characterization of milk protein nuclear RNAs in rat mammary gland. Anal Biochem 153: 211-20.
Busch H, Hnilica LS, Chien SC, Davis JR, Taylor CW. 1962. Isolation and purification of RP2-L, a nuclear protein fraction of the Walker 256 carcinosarcoma. Cancer Res 22: 637-45.
Cao Y, Liu R, Jiang X, Lu J, Jiang J, et al. 2009. Nuclear-cytoplasmic shuttling of menin regulates nuclear translocation of {beta}-catenin. Mol Cell Biol 29: 5477-87.
G, Favini E, Degl'Innocenti D, Salvi A, De Petro G, et al. 2009. RET/PTC1-driven neoplastic transformation and proinvasive phenotype of human thyrocytes involve Met induction and beta-catenin nuclear translocation. Neoplasia 11: 10-21.
Chaitanya GV, Babu PP. 2008. Multiple apoptogenic proteins are involved in the nuclear translocation of Apoptosis Inducing Factor during transient focal cerebral ischemia in rat. Brain Res 1246: 178-90.
Shipping and Storage: Ships at ambient temperature. Store at -20oC upon receipt.
Safety: Irritant. Avoid contact. Ships at ambient temperature. Storage on arrival: -20oC.
kw. nuclear protein isolation, nuclear protein extraction, nuclear protien - cytoplasmic protein fractionation, nuclear - cytoplasmic protein separation kit
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