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G-Actin/F-actin比值分析試劑盒,揭示細胞穩態變化

發布者:艾美捷科技    發布時間:2021-07-09     
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肌動蛋白.jpg

 

肌動蛋白是一類形成微絲的球狀多功能蛋白質 。它基本上存在于所有真核細胞中 ,進化過程中高度保守。細胞肌動蛋白以單體和聚合體形式存在于真核細胞內,其中單體小球又稱為G-肌動蛋白(Globular Actin,G-actin);多個G-肌動蛋白以雙螺旋結構聚合形成纖維化肌動蛋白,稱為F-肌動蛋白(Fibros Actin,F-actin),又稱為微絲,是細胞骨架最主要的結構組分之一。

 

G-Actin/F-Actin這2種形式在一定生理條件下相互轉換,在微絲正極組裝和負極去組裝同時存在的現象,此現象被命名為“踏車現象”(TreadMilling),并在細胞內保持平衡狀態,參與細胞一系列的生理功能。當G-Actin/F-Actin在細胞內比值發生改變時,意味著細胞結構,運動形態和穩態的變化。

 

因此G-Actin/F-Actin比值的檢測,常被用于:

1.研究藥物化合物對細胞穩態的影響。

2.研究突變的細胞系與野生型細胞的變化。

3.研究環境的變化對細胞的應激。

 

作為專業的生命科學醫藥原料供應商,CytoSkeleton中國區金牌代理,艾美捷科技為您推薦:G-Actin/F-Actin比值分析試劑盒( G-Actin:F-Actin 細胞體內分析試劑盒 )

名稱G-Actin/F-Actin比值分析試劑盒
G-Actin : F-Actin In Vivo Assay Kit
貨號CSK-BK037
品牌

CytoSkeleton.png

說明書下載點擊下載
原理? Western blot定量分析F-actin和G-actin比值:
細胞在以洗滌劑為基礎的裂解緩沖液中被裂解,該緩沖液能穩定和維持G型和F型細胞肌動蛋白。F-形式的細胞肌動蛋白。該緩沖液可溶解G型肌動蛋白,但不會溶解F型肌動蛋白。離心步驟使F-肌動蛋白顆?;?,將G-肌動蛋白留在上清液中。樣品上清液和顆粒在SDS-PAGE系統中運行,通過WB分析對肌動蛋白進行定量。
適用樣品懸浮細胞,貼壁細胞,組織樣本
試劑盒組分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
保存條件參考產品說明書,按照要求保存各組分

* 本產品僅適用于科研用途.

 

常見問題FAQ:

 

  1. G-Actin/F-Actin比值分析試劑盒的實驗中,進行到哪一步,可暫停實驗?

直到在37°C下以100,000xg旋轉1小時后才能停止測定。高速離心后,可以將上清液(G-肌動蛋白)與SDS上樣緩沖液混合并冷凍以備后用。沉淀物(F-肌動蛋白)應該用解聚劑和水重新懸浮,然后與SDS上樣緩沖液混合并冷凍以備后用。冷凍后,F-肌動蛋白解聚,因此有必要在冷凍樣品之前將F-肌動蛋白與G-肌動蛋白分離,以分離樣品以準確測量F-肌動蛋白和G-肌動蛋白的比率。

 

  1. G-Actin/F-Actin比值分析試劑盒的靈敏度多高?

該測定可以檢測到G-肌動蛋白與F-肌動蛋白比率的小至15%的變化。每個條件都應一式兩份進行并重復多次,因為實驗之間的檢測重現性可能會有10-20%的差異。

 

  1. G-Actin/F-Actin比值分析試劑盒能否在較慢的離心速度(如16,000g)下工作?

抱歉不行。在我們的測試離心速度是發現,如果低于100,000xg,哪怕是26,000 g,未能有效地沉淀F-肌動蛋白。

 

結果展示:

CytoSkeleton廠家質檢測試.jpg

Cul3+/+ 小鼠與Cul3+/? 小鼠的比較.png

3T3 swiss小鼠胚胎成纖維細胞系:
P=不可溶的F-actin;S=可溶性的G-actin
Panel 1 : 正常未處理細胞
Panel 2:肌動蛋白聚合藥物jasplakinolide處理
G-actin/F-actin ≈ 45/55
Cul3+/+ 小鼠與Cul3+/? 小鼠的比較:
G-actin/F-actin ≈ 45/55
CytoSkeleton廠家質檢測試Nature communications
doi.org/10.1038/s41467-021-23123-x

*以上結果僅供參考,因細胞系的不同,細胞所處的運動狀態,藥物與環境刺激不同,G-actin/F-actin的比值會有較大的浮動,建議參考相同細胞系的已發表文獻。

 

部分發表文章展示:

  • 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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