Revision 1

#12666Store at -20C

1 Kit

(6 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
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
EEA1 (C45B10) Rabbit mAb 3288 20 µl 170 kDa Rabbit IgG
Rab5A (E6N8S) Mouse mAb 46449 20 µl 25 kDa Mouse IgG1
Rab7 (D95F2) XP® Rabbit mAb 9367 20 µl 23 kDa Rabbit IgG
Rab11 (D4F5) XP® Rabbit mAb 5589 20 µl 25 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 Endosomal Marker Antibody Sampler Kit provides an economical means of distinguishing endosomes in the early, late, and recycling phases. The kit includes enough antibody 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 antibody.

Background

Endosomes are formed by the invagination of the plasma membrane to form vesicles in an effort to recycle components of the cell (1). Endosomes can be coated in clathrin when vesicles form at clathrin-coated pits (2). Caveolins are 21-24 kDa integral proteins that interact with cholesterol and are the main structural components of the cholesterol/sphingolipid-enriched plasma membrane caveolae (3). Each stage of endosome maturation is marked by a unique set of proteins. EEA1 is an early endosome marker that is essential for membrane fusion and trafficking (4). Members of the ras superfamily of small Rab GTPases, specifically Rab5, Rab7, and Rab11 are markers of the early, late and recycling endosomes (5).

  1. Huotari, J. and Helenius, A. (2011) EMBO J 30, 3481-500.
  2. Rodriguez-Boulan, E. et al. (2005) Nat Rev Mol Cell Biol 6, 233-47.
  3. Okamoto, T. et al. (1998) J Biol Chem 273, 5419-22.
  4. Christoforidis, S. et al. (1999) Nature 397, 621-5.
  5. 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.

    Limited Uses

    Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

    Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

    Revision 1
    #12666

    Endosomal Marker Antibody Sampler Kit

    Endosomal Marker 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).
    Endosomal Marker 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).
    Endosomal Marker 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).
    Endosomal Marker Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from various cell types using Caveolin-1 (D46G3) XP® Rabbit mAb.
    Endosomal Marker Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from various cell lines using EEA1 (C45B10) Rabbit mAb.
    Endosomal Marker Antibody Sampler Kit: Image 6 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).
    Endosomal Marker Antibody Sampler Kit: Image 7 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).
    Endosomal Marker Antibody Sampler Kit: Image 8 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).
    Endosomal Marker Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from various cell lines using Rab5A (E6N8S) Mouse mAb.
    Endosomal Marker Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from various cell lines using Clathrin Heavy Chain (D3C6) XP® Rabbit mAb.
    Endosomal Marker Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis of extracts from various cell lines using Rab11 (D4F5) XP® Rabbit mAb.
    Endosomal Marker Antibody Sampler Kit: Image 12 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.
    Endosomal Marker Antibody Sampler Kit: Image 13 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.
    Endosomal Marker Antibody Sampler Kit: Image 14 Expand Image
    Western blot analysis of extracts from various cell lines using Rab7 (D95F2) XP® Rabbit mAb.
    Endosomal Marker Antibody Sampler Kit: Image 15 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.
    Endosomal Marker Antibody Sampler Kit: Image 16 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).
    Endosomal Marker Antibody Sampler Kit: Image 17 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.
    Endosomal Marker Antibody Sampler Kit: Image 18 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).
    Endosomal Marker Antibody Sampler Kit: Image 19 Expand Image
    Confocal immunofluorescent analysis of A549 cells using Rab11 (D4F5) XP® Rabbit mAb (green). Actin filaments were labeled wtih DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Endosomal Marker Antibody Sampler Kit: Image 20 Expand Image
    Confocal immunofluorescent analysis of SK-MEL-28 cells using Rab7 (D95F2) XP® Rabbit mAb (green). Actin filaments were labeled using DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Endosomal Marker Antibody Sampler Kit: Image 21 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.
    Endosomal Marker Antibody Sampler Kit: Image 22 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.
    Endosomal Marker Antibody Sampler Kit: Image 23 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Caveolin-1 (D46G3) XP® Rabbit mAb.
    Endosomal Marker 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, and lane 3 is Rab5A (E6N8S) Mouse mAb. Western blot analysis was performed using Rab5 (C8B1) Rabbit mAb #3547.
    Endosomal Marker Antibody Sampler Kit: Image 25 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lymphoma using Caveolin-1 (D46G3) XP® Rabbit mAb.
    Endosomal Marker Antibody Sampler Kit: Image 26 Expand Image
    Immunohistochemical analysis of paraffin-embedded human ductal carcinoma of the breast using Rab5A (E6N8S) Mouse mAb.
    Endosomal Marker Antibody Sampler Kit: Image 27 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).
    Endosomal Marker Antibody Sampler Kit: Image 28 Expand Image
    Immunohistochemical analysis of paraffin-embedded human serous papillary carcinoma of the ovary using Rab5A (E6N8S) Mouse mAb.
    Endosomal Marker Antibody Sampler Kit: Image 29 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).
    Endosomal Marker Antibody Sampler Kit: Image 30 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).
    Endosomal Marker Antibody Sampler Kit: Image 31 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.
    Endosomal Marker Antibody Sampler Kit: Image 32 Expand Image
    Immunohistochemical analysis of paraffin-embedded human prostate adenocarcinoma using Rab5A (E6N8S) Mouse mAb.
    Endosomal Marker Antibody Sampler Kit: Image 33 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).