Revision 2

#80610Store at -20C

1 个试剂盒

(7 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

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For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
CD9 (D8O1A) Rabbit mAb 13174 20 µl 22, 24, 35 kDa Rabbit IgG
CD81 (D3N2D) Rabbit mAb 56039 20 µl 22 kDa Rabbit IgG
CD63 (E1W3T) Rabbit mAb 52090 20 µl 25-60 kDa Rabbit IgG
TSG101 (E6V1X) Rabbit mAb 72312 20 µl 50 kDa Rabbit IgG
Alix (E6P9B) Rabbit mAb 92880 20 µl 90-100 kDa Rabbit IgG
Flotillin-1 (D2V7J) XP® Rabbit mAb 18634 20 µl 49 kDa Rabbit IgG
Syntenin-1/MDA9 (E2I9L) Rabbit mAb 27964 20 µl 30 kDa Rabbit IgG
HSP70 Antibody 4872 20 µl 72, 73 kDa Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

The Human Reactive Exosome Marker Antibody Sampler Kit provides an economical means of analyzing proteins that can be present on exosomes. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/mL BSA, 50% glycerol, and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibodies.

Background

Exosomes are small (30-150 nm) membrane-bound vesicles that are secreted by various cell types under normal and pathological conditions (1,2). They originate from intracellular multivesicular endosomes upon fusion with the plasma membrane. Exosomes have emerged as an important mechanism of intercellular communication facilitating the transfer of membrane and cytosolic proteins, lipids, and RNA.

A variety of methods have been described to isolate exosomes and understand their composition (3-7). Heterogeneity in exosome composition can be attributed to the cells of origin as well as the isolation methods. However, there are protein markers that appear with high frequency. Tetraspanins are a family of cell surface glycoproteins with four transmembrane domains often found in exosomes (8). Tetraspanins CD9, CD81, and CD63 appear in exosomes and have been the target of immune-affinity approaches of exosome isolation. Flotillin-1 is a lipid raft-associated integral membrane protein that is incorporated into exosomes (9). Exosomes also contain proteins involved in endosomal membrane trafficking, collectively known as the ESCRT (endosomal sorting complex required for transport) pathway. Alix regulates cellular processes, such as endocytic membrane trafficking and cell adhesion through interactions with ESCRT proteins including endophilins, and CIN85 (Cbl-interacting protein of 85 kDa), and plays a role in exosome biogenesis (10-12). Syntenin-1 (MDA9, SDCBP) is a member of the PDZ family of proteins that functions as a scaffold adaptor protein regulating numerous signal transduction pathways (13). Syntenin-1 interacts with Alix to regulate exosome biogenesis (12). Tumor susceptibility gene 101 (TSG101) is a fundamental component of the ESCRT complex I involved in regulating the trafficking of proteins throughout the endosomal compartment (14). TSG101 is involved in regulating diverse biological processes, such as cell proliferation, viral budding and release, and exosome biosynthesis (15,16). The heat shock protein HSP70 is a molecular chaperone involved in protein folding that can be induced upon environmental stress (17). HSP70 may also be secreted through exosomes (18).

  1. Raposo, G. and Stoorvogel, W. (2013) J Cell Biol 200, 373-83.
  2. van Niel, G. et al. (2018) Nat Rev Mol Cell Biol 19, 213-228.
  3. Jeppesen, D.K. et al. (2019) Cell 177, 428-445.e18.
  4. Kowal, J. et al. (2016) Proc Natl Acad Sci U S A 113, E968-77.
  5. Sidhom, K. et al. (2020) Int J Mol Sci 21, 6466. doi: 10.3390/ijms21186466.
  6. Patel, G.K. et al. (2019) Sci Rep 9, 5335.
  7. Tauro, B.J. et al. (2012) Methods 56, 293-304.
  8. Hemler, M.E. (2005) Nat Rev Mol Cell Biol 6, 801-11.
  9. de Gassart, A. et al. (2003) Blood 102, 4336-44.
  10. Katoh, K. et al. (2003) J Biol Chem 278, 39104-13.
  11. Chatellard-Causse, C. et al. (2002) J Biol Chem 277, 29108-15.
  12. Baietti, M.F. et al. (2012) Nat Cell Biol 14, 677-85.
  13. Pradhan, A.K. et al. (2020) Cancer Metastasis Rev 39, 769-781.
  14. Katzmann, D.J. et al. (2001) Cell 106, 145-55.
  15. Garrus, J.E. et al. (2001) Cell 107, 55-65.
  16. Zhong, Q. et al. (1998) Cancer Res 58, 2699-702.
  17. Nollen, E.A. and Morimoto, R.I. (2002) J Cell Sci 115, 2809-16.
  18. Zhan, R. et al. (2009) Biochem Biophys Res Commun 387, 229-33.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    XP is a registered trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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    Revision 2
    #80610

    Human Reactive Exosome Marker Antibody Sampler Kit

    Human Reactive Exosome Marker Antibody Sampler Kit: Image 1 Expand Image
    Simple Western™ analysis of lysates (1.0 mg/mL) from HCT-116 cells using CD9 (D8O1A) Rabbit mAb #13174. 虚拟泳道式图像(左图)显示一抗稀释比例为 1:50 和 1:250 时的靶标条带(如图所示)。对应的电泳图(右图)为一抗稀释比例在 1:50(蓝线)和 1:250(绿线)时沿毛细血管内分子量的化学发光结果。在还原条件下,使用 12-230 kDa 分离模块在 ProteinSimple(BioTechne 品牌)的 Jess™ Simple Western 仪器上进行该实验。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 2 Expand Image
    使用 Syntenin-1/MDA9 (E2I9L) Rabbit mAb(上图)或 GAPDH (D16H11) XP® Rabbit mAb #5174(下图),对各种细胞系的提取物进行蛋白质印迹分析。Syntenin-1/MDA9 蛋白在 Daudi 细胞中阴性表达与预测的表达模式一致。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 3 Expand Image
    使用 Syntenin-1/MDA9 (E2I9L) Rabbit mAb(绿色)和 DAPI #4083 (蓝色),对 SK-MEL-5 细胞(左图,高表达)、A-172 细胞(中图,高表达)、MCF7 细胞(中图,低表达)或 Daudi 细胞(右图,阴性)进行共聚焦免疫荧光分析。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 4 Expand Image
    Flow cytometric analysis of Daudi cells (blue, negative) and MUTZ-3 cells (green, positive) using Syntenin-1/MDA9 (E2I9L) Rabbit mAb (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L)、F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 作为二抗。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 5 Expand Image
    使用 CD63 (E1W3T) Rabbit mAb(上图)、Myc-Tag (71D10) Rabbit mAb #2278(中图)或 β-Actin Antibody #4967(下图)对转染空载 (-) 或转染带 Myc/DDK 标签的全长人 CD63 蛋白 (hCD63-Myc/DDK;+) 表达载体的 CHO-K1 细胞提取物进行蛋白质印迹分析。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 6 Expand Image
    使用 CD9 (D8O1A) Rabbit mAb(上图)或 β-Actin (D6A8) Rabbit mAb #8457(下图)对不同细胞系提取物进行蛋白质印迹分析。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 7 Expand Image
    使用 Flotillin-1 (D2V7J) XP® Rabbit mAb 对不同细胞系提取物进行蛋白质印迹分析。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 8 Expand Image
    对 SK-MEL-5 细胞提取物的 syntenin-1/MDA9 蛋白进行免疫沉淀分析。泳道 1 为 10% 输入,泳道 2 为 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900,泳道 3 为 Syntenin-1/MDA9 (E2I9L) Rabbit mAb。使用 Syntenin-1/MDA9 (E2I9L) Rabbit mAb 进行蛋白质印迹分析。Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 用作二抗。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 9 Expand Image
    使用 HSP70 Antibody 对 HeLa、NIH/3T3、C6 和 COS 细胞提取物进行蛋白质印迹分析。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 10 Expand Image
    使用 CD63 (E1W3T) Rabbit mAb(上图)或 GAPDH (D16H11) XP® Rabbit mAb #5174(下图)对不同细胞系的提取物进行蛋白质印迹分析。CD63 蛋白在 Jurkat 和 MOLT-4 细胞中的低表达与预测的表达模式一致。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 11 Expand Image
    使用 CD81 (D3N2D) Rabbit mAb(上图)或 β-Actin (D6A8) Rabbit mAb #8457(下图)对不同细胞系的提取物进行蛋白印迹分析。U-937 细胞中 CD81 信号的缺失与已发表的报道一致。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 12 Expand Image

    使用 CD81 (D3N2D) Rabbit mAb 对石蜡包埋的人肝细胞癌组织进行免疫组织化学分析。

    Human Reactive Exosome Marker Antibody Sampler Kit: Image 13 Expand Image
    一抗与靶标蛋白结合之后,与偶联 HRP 的二抗形成复合体。添加 LumiGLO®,在酶催化分解期间发光。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 14 Expand Image
    使用 TSG101 (E6V1X) Rabbit mAb 对各种细胞系的提取物进行蛋白印迹分析。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 15 Expand Image
    使用 Alix (E6P9B) Rabbit mAb(上图)或 β-Actin (D6A8) Rabbit mAb #8457(下图)对 HCT 116 和 HCT 116 Alix 敲除型 (-/-) 细胞的提取物进行蛋白质印迹分析。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 16 Expand Image
    使用 CD9 (D8O1A) Rabbit mAb 对转染空载 (-) 或转染带 Myc/DDK 标签的全长人 CD9 蛋白 (hCD9-Myc/DDK; +) 表达载体的 293T 细胞提取物进行蛋白质印迹分析。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 17 Expand Image
    对 BT-20 细胞提取物的 flotillin-1 蛋白进行免疫沉淀。泳道 1 为 10% input,泳道 2为 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900,而泳道 3 为 Flotillin-1 (D2V7J) XP® Rabbit mAb。使用 Flotillin-1 (D2V7J) XP® Rabbit mAb 进行蛋白质印迹分析。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 18 Expand Image
    使用 HSP70 Antibody 对石蜡包埋的人乳腺癌细胞进行免疫组织化学分析。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 19 Expand Image

    使用 CD81 (D3N2D) Rabbit mAb 对石蜡包埋的人结肠腺癌组织进行免疫组织化学分析。

    Human Reactive Exosome Marker Antibody Sampler Kit: Image 20 Expand Image
    使用 TSG101 (E6V1X) Rabbit mAb(上图)或 β-Actin (D6A8) Rabbit mAb #8457(下图),对经对照 siRNA (-) 或 TSG101 siRNA (+) 转染的 293T 细胞的提取物进行蛋白印迹分析。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 21 Expand Image
    使用 Alix (E6P9B) Rabbit mAb 对不同细胞系的提取物进行蛋白质印迹分析。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 22 Expand Image
    使用 HSP70 Antibody 对石蜡包埋的人结肠癌细胞进行免疫组织化学分析。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 23 Expand Image
    使用 CD81 (D3N2D) Rabbit mAb 对石蜡包埋的人鳞状细胞癌组织进行免疫组织化学分析。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 24 Expand Image
    使用 TSG101 (E6V1X) Rabbit mAb(上图)、DYKDDDDK Tag (D6W5B) Rabbit mAb #14793(中间)或 β-Actin (D6A8) Rabbit mAb #8457(下图),对空载转染 (-) 或经表达 Myc/DDK 标记的全长人 TSG101(hTSG101-Myc/DDK;+)的构建体转染的 293T 细胞的提取物进行蛋白印迹分析。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 25 Expand Image
    对 K-562 细胞提取物 Alix 进行免疫沉淀。泳道 1 为 10% input,泳道 2 为 Rabbit (DA1E) mAb IgG XP® Isotype Control,泳道 3 为 Alix (E6P9B) Rabbit mAb。使用 Alix (E6P9B) Rabbit mAb 进行蛋白质印迹实验。Anti-rabbit IgG, HRP-linked Antibody #7074 用作二抗。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 26 Expand Image
    在对照肽(左图)或抗原特异性肽(右图)存在的情况下,使用 Flotillin-1 (D2V7J) XP® Rabbit mAb 对石蜡包埋的人肝细胞性肝癌进行免疫组织化学分析。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 27 Expand Image
    使用 HSP70 Antibody 对石蜡包埋的人肺癌细胞进行免疫组织化学分析,显示其定位于细胞浆。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 28 Expand Image

    使用 CD81 (D3N2D) Rabbit mAb 对石蜡包埋的人浆液性乳头状癌组织进行免疫组织化学分析。

    Human Reactive Exosome Marker Antibody Sampler Kit: Image 29 Expand Image
    使用 Alix (E6P9B) Rabbit mAb(绿色)对 HCT 116 细胞(左图,阳性)和 HCT 116 Alix 敲除型 (-/-) 细胞(右图,阴性)进行共聚焦免疫荧光分析。蓝色伪彩 = DRAQ5® #4084(DNA 荧光染料)。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 30 Expand Image
    使用 HSP70 Antibody 对石蜡包埋的人非霍奇金淋巴瘤细胞进行免疫组织化学分析。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 31 Expand Image

    使用 CD81 (D3N2D) Rabbit mAb 对石蜡包埋的人套细胞淋巴瘤组织进行免疫组织化学分析。

    Human Reactive Exosome Marker Antibody Sampler Kit: Image 32 Expand Image
    使用 Flotillin-1 (D2V7J) XP® Rabbit mAb(绿色)对 BT-20 细胞进行共聚焦免疫荧光分析。蓝色伪彩 = DRAQ5® #4084(DNA 荧光染料)。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 33 Expand Image
    使用 HSP70 Antibody 对石蜡包埋的人前列腺癌细胞进行免疫组织化学分析。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 34 Expand Image

    使用 CD81 (D3N2D) Rabbit mAb 对石蜡包埋的人正常肝脏组织进行免疫组织化学分析。

    Human Reactive Exosome Marker Antibody Sampler Kit: Image 35 Expand Image

    使用 CD81 (D3N2D) Rabbit mAb 对石蜡包埋的人正常小肠组织进行免疫组织化学分析。

    Human Reactive Exosome Marker Antibody Sampler Kit: Image 36 Expand Image
    使用 CD81 (D3N2D) Rabbit mAb 对石蜡包埋的正常人肾脏组织进行免疫组织化学分析。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 37 Expand Image

    使用 CD81 (D3N2D) Rabbit mAb 对石蜡包埋的人正常脑组织进行免疫组织化学分析。

    Human Reactive Exosome Marker Antibody Sampler Kit: Image 38 Expand Image
    与浓度匹配的 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(右图)相比,使用 CD81 (D3N2D) Rabbit mAb(左图)对石蜡包埋的人正常肝脏组织进行免疫组织化学分析。
    Human Reactive Exosome Marker Antibody Sampler Kit: Image 39 Expand Image
    使用 CD81 (D3N2D) Rabbit mAb 对石蜡包埋的 U-937 细胞沉淀物(左图,阳性)或 RL 细胞沉淀物(右图,阴性)进行免疫组织化学分析。