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Exo-Urine™ EV Isolation Kit 让你从尿液中发现更多biomarkers
发布时间:2020-01-09 12:20:25   [返回]
克服传统尿液外泌体提取方式的不足,获得高纯度高产量的尿液外泌体
从尿液中提取更多外泌体,从这个无创外泌体资源中发现更多biomarkers
使用SBI’s Exo-Urine EV Isolation Kit可以解决从尿液中提取外泌体纯度和产量面临的问题,尿液中存在着大量的糖蛋白,由尺寸排阻色谱法和特殊溶解试剂组成的Exo-Urine EV Isolation Kit 可以使外泌体从Tamm-Horstall protein (THP/uromodulin)聚合成的网状结构中逃脱,从而提取出高纯度高产量的外泌体。处理样品可以是少至1.5ml的新鲜尿液或冻存的尿液,使用Exo-Urine EV Isolation Kit 可以让从外泌体含量很低的尿液中,更好更有效的提取biomarkers
尿液中的外泌体为肾和泌尿系统相关的疾病提供丰富的biomarker,这些疾病包括急性肾损伤、泌尿生殖器癌症、慢性肾病、肾小球疾病、肾小管疾病以及肾移植排斥。因为尿液收集方便无创伤,从尿液中寻找biomarkers替代血清和血浆成为趋势。
受限于传统外泌体分离方法,外泌体提取遇到很多阻碍。特别是THP大量存在的情况下,这种蛋白可以包围EVs的网格,从尿液中很难得到干净浓度又高的外泌体。SBIs Exo-Urine EV Isolation Kit 让这些问题迎刃而解,让从尿液中获得biomarker变得更容易。
 高纯度:相对于多聚物沉淀和超速离心,去除更多的杂质蛋白,获取高浓度的外泌体。
 高产量:节约珍贵样品,外泌体产量相当于处理样本体积的1/10
 快速:处理10份样品比Exo-Urines fast-flow resin/gravity column setup的方法快了10个小时。
 灵活:无论新鲜尿液或者冻存样品都能获得产量和纯度相似的外泌体。
 灵敏:减少杂质,提高外泌体biomarker的浓度

* As assessed by western blotting for THP/uromodulin using equal volume loading.
** As assessed by normalized protein abundance using mass spectrometry.
*** EV yields from either 1.5 mL of urine (processed using the Exo-Urine Kit) or 15 mL of urine (processed using ultracentrifugation or polymer precipitation) were measured using ZetaView NTA and fNTA.

Reference

  1. Huebner AR, et al. Exosomes in urine biomarker discovery. Adv Exp Med Biol. 2015; 845:43-58. PMID: 25355568.

Supporting Data

See the superiority of EV isolation using the Exo-Urine EV Isolation Kit

Using fresh urine samples, the Exo-Urine Kit delivers preps that are enriched for EV-associated markers—with more CD63 than a competitor’s kit—and are low in contaminating THP]

Figure 1. Using fresh urine samples, the Exo-Urine Kit delivers preps that are enriched for EV-associated markers—with more CD63 than a competitor’s kit—and are low in contaminating THP. Freshly collected urine samples from three healthy donors were pooled and stored for 2, 4, or 6 hrs at 4°C before EV isolation and analysis via Western blot. (A) EV preps isolated from these samples using the Exo-Urine EV Isolation Kit show higher levels of the exosome-specific marker CD63 than EV preps isolated using Company N’s kit, and both EV isolation methods contained low levels of contaminating THP. Note that only 1.5 mL of the pooled urine was used for EV isolation with the Exo-Urine Kit, whereas 15 mL of pooled urine was needed for EV isolation using Company N’s kit. (B) EVs prepared using the Exo-Urine Kit are also enriched for the EV markers AQP2, TSG101, and HSP70.

Using frozen urine samples, the Exo-Urine Kit delivers EV preps that show greater enrichment of EV markers and less contaminating THP than other methods


Figure 2. Using frozen urine samples, the Exo-Urine Kit delivers EV preps that show greater enrichment of EV markers and less contaminating THP than other methods. Urine samples from healthy donors were pooled and stored at -20°C for 3 weeks before EV isolation and Western blotting for EV markers and contaminating THP. The superiority of the Exo-Urine Kit compared to other methods—Company N’s kit, ultracentrifugation, and polymer precipitation—is reflected in the higher levels of EV markers and almost complete lack of contaminating THP in the EVs isolated using the Exo-Urine Kit. As with Figure 1, only 1.5 mL of the pooled urine sample was used for the Exo-Urine prep compared to 15 mL for all other methods. High relative amounts of aquaporin-2 (AQP2) vs other EV markers in the ultracentrifugation and polymer precipitation lanes could result from carryover of non-EV associated forms of aquaporin-2 (Kodaka K, et al. Urinary Exosomal Aquaporin-2 Expression and the Efficacy of Tolvaptan in Advanced CKD Patients with Congestive Heart Failure: An Interim Report. J Clin Exp Nephrol. 2018 April. 3:6. DOI: 10.21767/2472-5056.100057).

The Exo-Urine Kit delivers higher yields of EVs (protein equivalent) than other methods


Figure 3. The Exo-Urine Kit delivers higher yields of EVs (protein equivalent) than other methods. EV preps from Figure 2 were analyzed for EV yield (as measured by protein equivalence using Qubit protein assay) and amounts normalized to the EVs isolated by ultracentrifugation. Use of the Exo-Urine Kit results in more EVs than all other methods.

EVs isolated using the Exo-Urine Kit possess typical EV morphology and low levels of filaments


Figure 4. EVs isolated using the Exo-Urine Kit possess typical EV morphology and low levels of filaments. Transmission electron microscopy (TEM) of EVs isolated from urine using either the Exo-Urine Kit, Company N’s kit, ultracentrifugation, or polymer precipitation show that the EVs isolated using the Exo-Urine Kit have the typical EV morphology and relatively fewer THP filaments than the other methods.

Mass spectrometry analysis of EVs isolated using the Exo-Urine Kit verify enrichment of EV markers and depletion of contaminating THPs


Figure 5. Mass spectrometry analysis of EVs isolated using the Exo-Urine Kit verify enrichment of EV markers and depletion of contaminating THP. LC-MS/MS analysis of the samples used in Figure 2 verify the Western blot analysis shown in Figure 2—EVs isolated using the Exo-Urine Kit show higher levels of EV markers, here Hsp70 and Anxa2—and lower levels of contaminating THP than EVs isolated using Company N’s kit, ultracentrifugation, or polymer precipitation.


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