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9765
Vesicle Trafficking Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Vesicle Trafficking Antibody Sampler Kit #9765

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Vesicle Trafficking Antibody Sampler Kit: Image 1

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 2

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 3

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 4

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 5

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 6

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 7

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).

Vesicle Trafficking Antibody Sampler Kit: Image 8

Western blot analysis of extracts from various cell lines using GOPC (D10A12) Rabbit mAb.

Vesicle Trafficking Antibody Sampler Kit: Image 9

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 10

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 11

Western blot analysis of extracts from various cell lines using Clathrin Heavy Chain (D3C6) XP® Rabbit mAb.

Vesicle Trafficking Antibody Sampler Kit: Image 12

Western blot analysis of extracts from various cell types using APPL1 (D83H4) XP® Rabbit mAb.

Vesicle Trafficking Antibody Sampler Kit: Image 13

Western blot analysis of extracts from various cell lines using EEA1 (C45B10) Rabbit mAb.

Vesicle Trafficking Antibody Sampler Kit: Image 14

Western blot analysis of extracts from various cell lines using Syntaxin 6 (C34B2) Rabbit mAb.

Vesicle Trafficking Antibody Sampler Kit: Image 15

Immunohistochemical analysis of paraffin-embedded human ductal carcinoma of the breast using Rab5A (E6N8S) Mouse mAb.

Vesicle Trafficking Antibody Sampler Kit: Image 16

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Caveolin-1 (D46G3) XP® Rabbit mAb.

Vesicle Trafficking Antibody Sampler Kit: Image 17

Immunohistochemical analysis of paraffin-embedded human serous papillary carcinoma of the ovary using Rab5A (E6N8S) Mouse mAb.

Vesicle Trafficking Antibody Sampler Kit: Image 18

Immunohistochemical analysis of paraffin-embedded human lymphoma using Caveolin-1 (D46G3) XP® Rabbit mAb.

Vesicle Trafficking Antibody Sampler Kit: Image 19

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 20

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 21

Immunohistochemical analysis of paraffin-embedded human prostate adenocarcinoma using Rab5A (E6N8S) Mouse mAb.

Vesicle Trafficking Antibody Sampler Kit: Image 22

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 23

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 24

Western blot analysis of extracts from various cell types using Caveolin-1 (D46G3) XP® Rabbit mAb.

Vesicle Trafficking Antibody Sampler Kit: Image 25

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 26

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 27

Western blot analysis of extracts from various cell lines using Rab5A (E6N8S) Mouse mAb.

To Purchase # 9765T
Product # Size Price
9765T
1 Kit  (8 x 20 µl) N/A

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-Caveolin-1 (Tyr14) Antibody 3251 20 µl
  • WB
H M R Mk 23, 25 Rabbit 
Caveolin-1 (D46G3) XP® Rabbit mAb 3267 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H M R Hm Mk B Dg 21, 24 Rabbit IgG
Clathrin Heavy Chain (D3C6) XP® Rabbit mAb 4796 20 µl
  • WB
  • IP
  • IF
H M R Mk 190 Rabbit IgG
APPL1 (D83H4) XP® Rabbit mAb 3858 20 µl
  • WB
  • IP
  • IF
H M R Mk 82 Rabbit IgG
EEA1 (C45B10) Rabbit mAb 3288 20 µl
  • WB
  • IP
  • IF
H M R 170 Rabbit IgG
Syntaxin 6 (C34B2) Rabbit mAb 2869 20 µl
  • WB
  • IP
  • IF
H M R 32 Rabbit IgG
Rab5A (E6N8S) Mouse mAb 46449 20 µl
  • WB
  • IP
  • IHC
  • IF
H M R Mk 25 Mouse IgG1
GOPC (D10A12) Rabbit mAb 8576 20 µl
  • WB
H 59 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl
  • WB
Horse 

Product 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.

Specificity / Sensitivity

Phospho-Caveolin-1 (Tyr14) Antibody detects endogenous levels of Caveolin-1 only when phosphorylated at Tyr14 and does not cross-react with caveolin-2, -3 or caveolin-1β, the short isoform of caveolin-1. Caveolin-1 (D46G3) XP® Rabbit mAb detects endogenous levels of total caveolin-1 protein. Clathrin Heavy Chain (D3C6) XP® Rabbit mAb detects endogenous levels of total clathrin protein. APPL1 (D83H4) XP® Rabbit mAb detects endogenous levels of total APPL1 protein. EEA1 (C45B10) Rabbit mAb detects endogenous levels of total EEA1 protein. Syntaxin 6 (C34B2) Rabbit mAb detects endogenous levels of total Syntaxin 6 protein. GOPC (D10A12) Rabbit mAb detects endogenous levels of total GOPC protein. Rab5A (E6N8S) Mouse mAb detects endogenous levels of total Rab5A protein.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Tyr14 of human caveolin-1. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Glu20 of human caveolin-1, Pro1663 of human clathrin heavy chain protein, Thr426 of human APPL1, Ser70 of human EEA1 protein, Tyr140 of mouse syntaxin 6 protein, Phe31 of human GOPC protein, and Gly190 of human Rab5A protein.

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.

Limited Uses

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For Research Use Only. Not For Use In Diagnostic Procedures.
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.
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