Revision 1

#9765Store at -20C

1 Kit

(8 x 20 microliters)

Cell Signaling Technology

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

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Phospho-Caveolin-1 (Tyr14) Antibody 3251 20 µl 23, 25 kDa Rabbit 
Caveolin-1 (D46G3) XP® Rabbit mAb 3267 20 µl 21, 24 kDa Rabbit IgG
Clathrin Heavy Chain (D3C6) XP® Rabbit mAb 4796 20 µl 190 kDa Rabbit IgG
APPL1 (D83H4) XP® Rabbit mAb 3858 20 µl 82 kDa Rabbit IgG
EEA1 (C45B10) Rabbit mAb 3288 20 µl 170 kDa Rabbit IgG
Syntaxin 6 (C34B2) Rabbit mAb 2869 20 µl 32 kDa Rabbit IgG
Rab5A (E6N8S) Mouse mAb 46449 20 µl 25 kDa Mouse IgG1
GOPC (D10A12) Rabbit mAb 8576 20 µl 59 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl Horse 

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

Description

The Vesicle Trafficking Antibody Sampler kit provides an economical means to analyze proteins involved in the intracellular transport of cargo proteins. This kit includes enough primary and secondary antibody to perform two western blot experiments.

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 antibody.

Background

Vesicle trafficking is an integral cellular process and the associated proteins involved also play major roles in other signaling pathways. Caveolins are involved in diverse biological functions including vesicular trafficking, cholesterol homeostasis, cell adhesion, apoptosis, and are also indicated in neurodegenerative disease (1). It is believed that caveolins serve as scaffolding proteins for the integration of signal transduction. Phosphorylation at Tyr14 is essential for caveolin association with SH2 or PTB domain-containing adaptor proteins, such as GRB7 (2-4).

Clathrin-coated vesicles provide for the intracellular transport of proteins following endocytosis and during multiple vesicle trafficking pathways. Vesicles form at specialized areas of the cell membrane where clathrin and associated proteins form clathrin-coated pits. Invagination of these cell membrane-associated pits internalizes proteins and forms an intracellular clathrin-coated vesicle (5,6). Clathrin is the most abundant protein in these vesicles and is present as a basic assembly unit called a triskelion. Each clathrin triskelion is composed of three clathrin heavy chains and three clathrin light chains. Clathrin heavy chain proteins are composed of several functional domains that associate with other vesicle proteins (6).

The APPL1 multidomain adaptor protein is a BAR-domain protein family member that is involved in membrane trafficking within a number of signal transduction pathways (7).

EEA1 is an early endosomal marker and a Rab5 effector protein essential for early endosomal membrane fusion and trafficking (8,9). Syntaxin 6 is a ubiquitously expressed S25C family member of the SNARE proteins (10,11). Syntaxin 6 protein is localized to the trans-Golgi and within endosomes and regulates membrane trafficking by partnering with a variety of other SNARE proteins (12-14). It has two coiled-coil domains (CC1 and CC2) located in the amino-terminal region and a PDZ domain in the carboxy-terminal region (15). The CC2 domain and its adjacent linker region mediate the association of GOPC with the Golgi protein golgin-160 and the Q-SNARE protein syntaxin 6 (15,16). The PDZ domain of GOPC interacts with the carboxy terminus of target proteins to mediate target protein vesicular trafficking and surface expression (17-20).

Rab5 is a member of the Ras superfamily of small Rab GTPases. Rab5 is localized at the plasma membrane and early endosomes and functions as a key regulator of vesicular trafficking during early endocytosis (21).

  1. Smart, E.J. et al. (1999) Mol Cell Biol 19, 7289-304.
  2. Nomura, R. and Fujimoto, T. (1999) Mol Biol Cell 10, 975-86.
  3. Volonté, D. et al. (2001) J Biol Chem 276, 8094-103.
  4. Lee, H. et al. (2000) Mol Endocrinol 14, 1750-75.
  5. Rodriguez-Boulan, E. et al. (2005) Nat Rev Mol Cell Biol 6, 233-47.
  6. Mousavi, S.A. et al. (2004) Biochem J 377, 1-16.
  7. Habermann, B. (2004) EMBO Rep 5, 250-5.
  8. Mu, F.T. et al. (1995) J Biol Chem 270, 13503-11.
  9. Christoforidis, S. et al. (1999) Nature 397, 621-5.
  10. Bock, J.B. et al. (2001) Nature 409, 839-41.
  11. Bock, J.B. et al. (1996) J Biol Chem 271, 17961-5.
  12. Wendler, F. and Tooze, S. (2001) Traffic 2, 606-11.
  13. Bock, J.B. et al. (1997) Mol Biol Cell 8, 1261-71.
  14. Mallard, F. et al. (2002) J Cell Biol 156, 653-64.
  15. Charest, A. et al. (2001) J Biol Chem 276, 29456-65.
  16. Hicks, S.W. and Machamer, C.E. (2005) J Biol Chem 280, 28944-51.
  17. Cheng, J. et al. (2002) J Biol Chem 277, 3520-9.
  18. He, J. et al. (2004) J Biol Chem 279, 50190-6.
  19. Wente, W. et al. (2005) J Biol Chem 280, 32419-25.
  20. Ito, H. et al. (2006) Biochem J 397, 389-98.
  21. Zerial, M. and McBride, H. (2001) Nat Rev Mol Cell Biol 2, 107-17.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

    使用に関する制限

    法的な権限を与えられたCSTの担当者が署名した書面によって別途明示的に合意された場合を除き、 CST、その関連会社または代理店が提供する製品には以下の条件が適用されます。お客様が定める条件でここに定められた条件に含まれるものを超えるもの、 または、ここに定められた条件と異なるものは、法的な権限を与えられたCSTの担当者が別途書面にて受諾した場合を除き、拒絶され、 いかなる効力も効果も有しません。

    研究専用 (For Research Use Only) またはこれに類似する表示がされた製品は、 いかなる目的についても FDA または外国もしくは国内のその他の規制機関により承認、認可または許可を受けていません。 お客様は製品を診断もしくは治療目的で使用してはならず、また、製品に表示された内容に違反する方法で使用してはなりません。 CST が販売または使用許諾する製品は、エンドユーザーであるお客様に対し、使途を研究および開発のみに限定して提供されるものです。 診断、予防もしくは治療目的で製品を使用することまたは製品を再販売 (単独であるか他の製品等の一部であるかを問いません) もしくはその他の商業的利用の目的で購入することについては、CST から別途許諾を得る必要があります。 お客様は以下の事項を遵守しなければなりません。(a) CST の製品 (単独であるか他の資材と一緒であるかを問いません) を販売、使用許諾、貸与、寄付もしくはその他の態様で第三者に譲渡したり使用させたりしてはなりません。また、商用の製品を製造するために CST の製品を使用してはなりません。(b) 複製、改変、リバースエンジニアリング、逆コンパイル、 分解または他の方法により製品の構造または技術を解明しようとしてはなりません。また、 CST の製品またはサービスと競合する製品またはサービスを開発する目的で CST の製品を使用してはなりません。(c) CST の製品の商標、商号、ロゴ、特許または著作権に関する通知または表示を除去したり改変したりしてはなりません。(d) CST の製品をCST 製品販売条件(CST’s Product Terms of Sale) および該当する書面のみに従って使用しなければなりません。(e) CST の製品に関連してお客様が使用する第三者の製品またはサービスに関する使用許諾条件、 サービス提供条件またはこれに類する合意事項を遵守しなければなりません。

    Revision 1
    #9765

    Vesicle Trafficking Antibody Sampler Kit

    Vesicle Trafficking Antibody Sampler Kit: Image 1 Expand Image
    Confocal immunofluorescent analysis of fixed frozen mouse pancreas labeled with Caveolin-1 (D46G3) XP® Rabbit mAb (left, red), and colabeled with DyLight 488 Phalloidin #12935 (right, green) and DAPI #4083 (right, blue).
    Vesicle Trafficking Antibody Sampler Kit: Image 2 Expand Image
    Confocal immunofluorescent analysis of fixed frozen mouse kidney labeled with Caveolin-1 (D46G3) XP® Rabbit mAb (left, red), and colabeled with DyLight 488 Phalloidin #12935 (right, green) and DAPI #4083 (right, blue).
    Vesicle Trafficking Antibody Sampler Kit: Image 3 Expand Image
    Confocal immunofluorescent analysis of fixed frozen mouse skeletal muscle labeled with Caveolin-1 (D46G3) XP® Rabbit mAb (left, red), and colabeled with DyLight 488 Phalloidin #12935 (right, green) and DAPI #4083 (right, blue).
    Vesicle Trafficking Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from various cell lines using Syntaxin 6 (C34B2) Rabbit mAb.
    Vesicle Trafficking Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from HeLa cells, untreated (-) or H2O2-treated (+), using Phospho-Caveolin-1 (Tyr14) Antibody (upper) and Caveolin-1 (D46G3) XP® Rabbit mAb #3267 (lower).
    Vesicle Trafficking Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from various cell types using Caveolin-1 (D46G3) XP® Rabbit mAb.
    Vesicle Trafficking Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from various cell lines using EEA1 (C45B10) Rabbit mAb.
    Vesicle Trafficking Antibody Sampler Kit: Image 8 Expand Image
    Confocal immunofluorescent tile scan of a fixed frozen brain from an amyloid mouse model of Alzheimer's disease using EEA1 (C45B10) Rabbit mAb #3288 (green), GFAP (GA5) Mouse mAb #3670 (red), and DAPI #4083 (blue).
    Vesicle Trafficking Antibody Sampler Kit: Image 9 Expand Image
    Confocal immunofluorescent analysis of fixed frozen mouse thalamus, labeled with EEA1 (C45B10) Rabbit mAb #3288 (left, green) and co-labeled with GFAP (GA5) Mouse mAb #3670 (right, red) and DAPI #4083 (right, blue).
    Vesicle Trafficking Antibody Sampler Kit: Image 10 Expand Image
    Confocal immunofluorescent analysis of fixed frozen mouse hippocampus, labeled with EEA1 (C45B10) Rabbit mAb #3288 (left, green) and co-labeled with GFAP (GA5) Mouse mAb #3670 (right, red) and DAPI #4093 (right, blue).
    Vesicle Trafficking Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis of extracts from various cell types using APPL1 (D83H4) XP® Rabbit mAb.
    Vesicle Trafficking Antibody Sampler Kit: Image 12 Expand Image
    Western blot analysis of extracts from various cell lines using Rab5A (E6N8S) Mouse mAb.
    Vesicle Trafficking Antibody Sampler Kit: Image 13 Expand Image
    Western blot analysis of extracts from various cell lines using Clathrin Heavy Chain (D3C6) XP® Rabbit mAb.
    Vesicle Trafficking Antibody Sampler Kit: Image 14 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
    Vesicle Trafficking Antibody Sampler Kit: Image 15 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO* is added and emits light during enzyme catalyzed decomposition.
    Vesicle Trafficking Antibody Sampler Kit: Image 16 Expand Image
    Western blot analysis of extracts from various cell lines using GOPC (D10A12) Rabbit mAb.
    Vesicle Trafficking Antibody Sampler Kit: Image 17 Expand Image
    Confocal immunofluorescent analysis of MCF7 cells using Syntaxin 6 (C34B2) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5 (fluorescent DNA dye).
    Vesicle Trafficking Antibody Sampler Kit: Image 18 Expand Image
    Western blot analysis of extracts from control HeLa cells (lane 1) or caveolin-1 knockout HeLa cells (lane 2) using Caveolin-1 (D46G3) XP® Rabbit mAb #3267 (upper), or β-actin (D6A8) Rabbit mAb #8457 (lower). The absence of signal in the caveolin-1 knockout HeLa cells confirms the specificity of the antibody to caveolin-1.
    Vesicle Trafficking Antibody Sampler Kit: Image 19 Expand Image
    Confocal immunofluorescent analysis of HeLa cells using EEA1 (C45B10) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5 (fluorescent DNA dye).
    Vesicle Trafficking Antibody Sampler Kit: Image 20 Expand Image
    Confocal immunofluorescent analysis of HeLa cells using APPL1 (D83H4) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Vesicle Trafficking Antibody Sampler Kit: Image 21 Expand Image
    Western blot analysis of HAP1 extracts from WT (left) or RAB5A KO (right) using Rab5A (E6N8S) Mouse mAb. Membranes stained with Ponceau S for total protein normalization (lower). These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies, as a companion to validation data generated by CST scientists.
    Vesicle Trafficking Antibody Sampler Kit: Image 22 Expand Image
    Confocal immunofluorescent analysis of SH-SY5Y cells using Clathrin Heavy Chain (D3C6) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Vesicle Trafficking Antibody Sampler Kit: Image 23 Expand Image
    Immunoprecipitation of caveolin-1 from HeLa cells using Caveolin-1 (D46G3) XP® Rabbit mAb followed by western blot using the same antibody. Lane 1 is 5% input.
    Vesicle Trafficking Antibody Sampler Kit: Image 24 Expand Image
    Immunoprecipitation of Rab5A protein from HeLa cell extracts. Lane 1 is 10% input, lane 2 is Mouse (G3A1) mAb IgG1 Isotype Control #5415, lane 3 is Rab5A (E6N8S) Mouse mAb without HeLa cell extracts, and lane 4 is Rab5A (E6N8S) Mouse mAb. Western blot analysis was performed using Rab5A (E6N8S) Mouse mAb.
    Vesicle Trafficking Antibody Sampler Kit: Image 25 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Caveolin-1 (D46G3) XP® Rabbit mAb.
    Vesicle Trafficking Antibody Sampler Kit: Image 26 Expand Image
    Immunoprecipitation of Rab5A protein from HeLa cell extracts. Lane 1 is 10% input, lane 2 is Mouse (G3A1) mAb IgG1 Isotype Control #5415, and lane 3 is Rab5A (E6N8S) Mouse mAb. Western blot analysis was performed using Rab5 (C8B1) Rabbit mAb #3547.
    Vesicle Trafficking Antibody Sampler Kit: Image 27 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lymphoma using Caveolin-1 (D46G3) XP® Rabbit mAb.
    Vesicle Trafficking Antibody Sampler Kit: Image 28 Expand Image
    Immunohistochemical analysis of paraffin-embedded human ductal carcinoma of the breast using Rab5A (E6N8S) Mouse mAb.
    Vesicle Trafficking Antibody Sampler Kit: Image 29 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse lung using Caveolin-1 (D46G3) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
    Vesicle Trafficking Antibody Sampler Kit: Image 30 Expand Image
    Immunohistochemical analysis of paraffin-embedded human serous papillary carcinoma of the ovary using Rab5A (E6N8S) Mouse mAb.
    Vesicle Trafficking Antibody Sampler Kit: Image 31 Expand Image
    Confocal immunofluorescent analysis of C2C12 cells using Caveolin-1 (D46G3) XP® Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® (fluorescent DNA dye).
    Vesicle Trafficking Antibody Sampler Kit: Image 32 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using Rab5A (E6N8S) Mouse mAb (left) compared to concentration matched Mouse (G3A1) IgG1 Isotype Control #5415 (right).
    Vesicle Trafficking Antibody Sampler Kit: Image 33 Expand Image
    Flow cytometric analysis of HeLa cells using Caveolin-1 (D46G3) XP® Rabbit mAb (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    Vesicle Trafficking Antibody Sampler Kit: Image 34 Expand Image
    Immunohistochemical analysis of paraffin-embedded human prostate adenocarcinoma using Rab5A (E6N8S) Mouse mAb.
    Vesicle Trafficking Antibody Sampler Kit: Image 35 Expand Image
    Confocal immunofluorescent analysis of HeLa cells using Rab5A (E6N8S) Mouse mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).