细胞稳态变化研究——G-Actin/F-actin比值分析试剂盒方案来啦

 

肌动蛋白

 

肌动蛋白是一类形成微丝的球状多功能蛋白质 。它基本上存在于所有真核细胞中 ,进化过程中高度保守。细胞肌动蛋白以单体和聚合体形式存在于真核细胞内,其中单体小球又称为G-肌动蛋白(Globular Actin,G-actin);多个G-肌动蛋白以双螺旋结构聚合形成纤维化肌动蛋白,称为F-肌动蛋白(Fibros Actin,F-actin),又称为微丝,是细胞骨架最主要的结构组分之一。

 

G-Actin/F-Actin这2种形式在一定生理条件下相互转换,在微丝正极组装和负极去组装同时存在的现象,此现象被命名为“踏车现象”(TreadMilling),并在细胞内保持平衡状态,参与细胞一系列的生理功能。当G-Actin/F-Actin在细胞内比值发生改变时,意味着细胞结构,运动形态和稳态的变化。

 

因此G-Actin/F-Actin比值的检测,常被用于:

1.研究药物化合物对细胞稳态的影响。

2.研究突变的细胞系与野生型细胞的变化。

3.研究环境的变化对细胞的应激。

 

作为专业的生命科学医药原料供应商,CytoSkeleton中国区金牌代理,艾美捷科技为您推荐:G-Actin/F-Actin比值分析试剂盒( G-Actin:F-Actin 细胞体内分析试剂盒 )

名称 G-Actin/F-Actin比值分析试剂盒
G-Actin : F-Actin In Vivo Assay Kit
货号 CSK-BK037
品牌 Cytoskeleton
说明书下载 点击下载
原理 Ø Western blot定量分析F-actin和G-actin比值:
细胞在以洗涤剂为基础的裂解缓冲液中被裂解,该缓冲液能稳定和维持G型和F型细胞肌动蛋白。F-形式的细胞肌动蛋白。该缓冲液可溶解G型肌动蛋白,但不会溶解F型肌动蛋白。离心步骤使F-肌动蛋白颗粒化,将G-肌动蛋白留在上清液中。样品上清液和颗粒在SDS-PAGE系统中运行,通过WB分析对肌动蛋白进行定量。
适用样品 悬浮细胞,贴壁细胞,组织样本
试剂盒组分 1. Lysis and F-actin stabilization buffer
2. ATP (Cat. # BSA04)
3. Protease inhibitor cocktail (Cat. # PIC02)
4. F-actin enhancing control solution
5. F-actin depolymerization control solution
6. Control G-actin Standard (Cat. # AKL99)
7. Anti-Actin MAb (clone 7A8.2.1) (Cat # AAN02-S)
8. SDS sample buffer (5 x)
9. DMSO
10. Manual with detailed protocols and extensive troubleshooting guide
保存条件 参考产品说明书,按照要求保存各组分

* 本产品仅适用于科研用途.

 

常见问题FAQ:

 

  1. G-Actin/F-Actin比值分析试剂盒的实验中,进行到哪一步,可暂停实验?

直到在37°C下以100,000xg旋转1小时后才能停止测定。高速离心后,可以将上清液(G-肌动蛋白)与SDS上样缓冲液混合并冷冻以备后用。沉淀物(F-肌动蛋白)应该用解聚剂和水重新悬浮,然后与SDS上样缓冲液混合并冷冻以备后用。冷冻后,F-肌动蛋白解聚,因此有必要在冷冻样品之前将F-肌动蛋白与G-肌动蛋白分离,以分离样品以准确测量F-肌动蛋白和G-肌动蛋白的比率。

 

  1. G-Actin/F-Actin比值分析试剂盒的灵敏度多高?

该测定可以检测到G-肌动蛋白与F-肌动蛋白比率的小至15%的变化。每个条件都应一式两份进行并重复多次,因为实验之间的检测重现性可能会有10-20%的差异。

 

  1. G-Actin/F-Actin比值分析试剂盒能否在较慢的离心速度(如16,000g)下工作?

抱歉不行。在我们的测试离心速度是发现,如果低于100,000xg,哪怕是26,000 g,未能有效地沉淀F-肌动蛋白。

 

结果展示:

3T3 swiss小鼠胚胎成纤维细胞系 Cul3+/+ 小鼠与Cul3+/− 小鼠的比较
3T3 swiss小鼠胚胎成纤维细胞系:
P=不可溶的F-actin;S=可溶性的G-actin
Panel 1 : 正常未处理细胞
Panel 2:肌动蛋白聚合药物jasplakinolide处理
G-actin/F-actin ≈ 45/55
Cul3+/+ 小鼠与Cul3+/− 小鼠的比较:
G-actin/F-actin ≈ 45/55
CytoSkeleton厂家质检测试 Nature communications
doi.org/10.1038/s41467-021-23123-x

*以上结果仅供参考,因细胞系的不同,细胞所处的运动状态,药物与环境刺激不同,G-actin/F-actin的比值会有较大的浮动,建议参考相同细胞系的已发表文献。

 

部分发表文章展示:

  • Morandell, Jasmin, et al. "Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain development." Nature communications1 (2021): 1-22.
  • Lu, Yi-Ju, et al. "Arabidopsis calcium-dependent protein kinase 3 regulates actin cytoskeleton organization and immunity." Nature communications1 (2020): 1-12.
  • Bojcevski, Jovana et al. “Influence of retinal NMDA receptor activity during autoimmune optic neuritis.” Journal of neurochemistry vol. 153,6 (2020): 693-709. doi:10.1111/jnc.14980
  • Chen, Y. et al.  "Coronin 2B regulates dendrite outgrowth by modulating actin dynamics." FEBS Lett, 594: 2975-2987. (2020). https://doi.org/10.1002/1873-3468.13886
  • Yang, N. et al. Effects of dexamethasone on remodeling of the hippocampal synaptic filamentous actin cytoskeleton in a model of pilocarpine-induced status epilepticus. Int. J. Med. Sci. 17, 1683–1691 (2020).
  • Gupta, R., Kumar, G., Jain, B. P., Chandra, S. & Goswami, S. K. Ectopic expression of 35 kDa and knocking down of 78 kDa SG2NAs induce cytoskeletal reorganization, alter membrane sialylation, and modulate the markers of EMT. Mol. Cell. Biochem. (2020) doi:10.1007/s11010-020-03932-2.
  • Musah, A. S. et al. Mechanistic Target of Rapamycin Regulates the Oligodendrocyte Cytoskeleton during Myelination. J. Neurosci. 40, 2993 LP – 3007 (2020).
  • Shi, Zengdun et al. “Myocardin and myocardin-related transcription factor-A synergistically mediate actin cytoskeletal-dependent inhibition of liver fibrogenesis.” American journal of physiology. Gastrointestinal and liver physiology vol. 318,3 (2020): G504-G517. doi:10.1152/ajpgi.00302.2019
  • Yang, Nuo et al. “Effects of Dexamethasone on Remodeling of the Hippocampal Synaptic Filamentous Actin Cytoskeleton in a Model of Pilocarpine-induced Status Epilepticus.” International journal of medical sciences vol. 17,12 1683-1691. 2 Jul. 2020, doi:10.7150/ijms.44927
  • Bojcevski J, Stojic A, Hoffmann DB, Williams SK, Müller A, Diem R, Fairless R. Influence of retinal NMDA receptor activity during autoimmune optic neuritis. J Neurochem. 2020 Jun;153(6):693-709. doi: 10.1111/jnc.14980. Epub 2020Mar 3. PMID: 32031240.
  • Kommaddi, Reddy Peera et al. “Aβ mediates F-actin disassembly in dendritic spines leading to cognitive deficits in Alzheimer's disease.” The Journal of neuroscience : the official journal of the Society for Neuroscience vol. 38,5 (2018): 1085-1099. doi:10.1523/JNEUROSCI.2127-17.2017
  • Kommaddi RP et al. Aβ mediates F-actin disassembly in dendritic spines leading to cognitive deficits in Alzheimer's disease. J Neurosci. 2018Jan 31;38(5):1085-1099. doi: 10.1523/JNEUROSCI.2127-17.2017. Epub 2017 Dec 15. PMID: 29246925; PMCID: PMC5792472.
  • Asahara et al., 2013. Ras-related C3 botulinum toxin substrate 1 (RAC1) regulates glucose-stimulated insulin secretion via modulation of F-actin. Diabetologia. doi: 10.1007/s00125-013-2849-5.
  • Ni et al., 2013. The role of RhoA and cytoskeleton in myofibroblast transformation in hyperoxic lung fibrosis. Free Radical Biology and Medicine. doi: http://dx.doi.org/10.1016/j.freeradbiomed.2013.03.012.
  • Ramachandran et al., 2013. JunB mediates basal- and TGFb1-induced smooth muscle cell contractility. PLoS ONE. 8(1): e53430.
  • Shuang et al., 2013. Destrin deletion enhances the bone loss in hindlimb suspended mice. E. J. Appl. Physiol. 113, 403-410.
  • Malenda et al., 2012. Statins Impair Glucose Uptake in Tumor Cells. Neoplasia. 14, 311–323.
  • Fan et al., 2012. A role for γS-crystallin in the organization of actin and fiber cell maturation in the mouse lens. FEBS. J. 279, 2892-2904.
  • Liu et al., 2012. TLR2 Is a Primary Receptor for Alzheimer's Amyloid β Peptide To Trigger Neuroinflammatory Activation. J. Immunol. 188, 1098-1107.
  • Chand et al., 2012. C-terminal region of teneurin-1 co-localizes with dystroglycan and modulates cytoskeletal organization through an extracellular signal-regulated kinase-dependent stathmin- and filamin A-mediated mechanism in hippocampal cells. Neuroscience. 219, 255-270.
  • Rapier et al., 2010. The extracellular matrix microtopography drives critical changes in cellular motility and Rho A activity in colon cancer cells. Cancer Cell Int. 10, 24.
  • Meeks et al., 2005. Heat shock protein 20-mediated force suppression in forskolin-relaxed swine carotid artery. Am. J. Physiol. 288, C633-C639.
  • Zhang et al., 2005. Activation of the Arp2/3 complex by N-WASP is required for actin polymerization and contraction in smooth muscle. Am. J. Physiol. 288, C1145-C1160.
  • Chen et al., 2004. Protective effect of phosphatidylinositol 4,5-bisphosphate against cortical filamentous actin loss and insulin resistance induced by sustained exposure of 3T3-L1 adipocytes to insulin. J. Biol. Chem. 279, 39705-39709.
  • Tang and Gunst, 2004. The small GTPase Cdc42 regulates actin polymerization and tension development during contractile stimulation of smooth muscle. J. Biol. Chem. 279, 51722-51728.
  • Searles et al., 2004. Actin cytoskeleton organization and poststranscriptional regulation of endothelial nitric oxide synthase during cell growth. Circ. Res. 95, 488-495.
  • Tu et al., 2003. Migfilin and Mig-2 link focal adhesions to filamin and the actin cytoskeleton and function in cell shape modulation. Cell. 113, 37-47.

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文章来源:G-Actin/F-actin比值分析试剂盒,揭示细胞稳态变化

 

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