Revision 1

#9778Store at -20C

 

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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
Raptor (24C12) Rabbit mAb 2280 40 µl 150 kDa Rabbit 
mTOR (7C10) Rabbit mAb 2983 40 µl 289 kDa Rabbit IgG
RagB (D18F3) Rabbit mAb 8150 40 µl 40 kDa Rabbit IgG
RagA (D8B5) Rabbit mAb 4357 40 µl 30 kDa Rabbit IgG
RagD Antibody 4470 40 µl 50 kDa Rabbit 
RagC (D31G9) XP® Rabbit mAb 5466 40 µl 50 kDa Rabbit IgG
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 Rag Protein Antibody Sampler Kit provides an economical means to evaluate the regulation of mTORC1 signaling by such proteins as Raptor, RagA, RagB, RagC, and RagD. The kit contains enough primary and secondary antibodies to perform four Western blot experiments per 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

The mTORC1 kinase complex is a critical regulator of cell growth (1,2). Its activity is modulated by energy levels, growth factors, and amino acids via signaling through Akt, MAPK, and AMPK pathways (3,4). Recent studies found that the four related GTPases, RagA, RagB, RagC, and RagD, interact with raptor within the mTORC1 complex (1,2). These interactions are both necessary and sufficient for mTORC1 activation in response to amino acid signals (1,2).

  1. Sancak, Y. et al. (2008) Science 320, 1496-501.
  2. Kim, E. et al. (2008) Nat Cell Biol 10, 935-45.
  3. Hay, N. and Sonenberg, N. (2004) Genes Dev 18, 1926-45.
  4. Wullschleger, S. et al. (2006) Cell 124, 471-84.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a 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 1
    #9778

    Rag Protein Antibody Sampler Kit

    Rag Protein Antibody Sampler Kit: Image 1 Expand Image
    Western blot analysis of extracts from various cell lines using mTOR (7C10) Rabbit mAb.
    Rag Protein Antibody Sampler Kit: Image 2 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from Hela cells using mTOR (7C10) Rabbit mAb #2983. The virtual lane view (left) shows a single target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 66-440 kDa separation module.
    Rag Protein Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from various cell lines, using Raptor (24C12) Rabbit mAb.
    Rag Protein Antibody Sampler Kit: Image 4 Expand Image
    Immunoprecipitation of mTOR protein from MCF-7 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is mTOR (7C10) Rabbit mAb. Western blot analysis was performed using mTOR (7C10) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.
    Rag Protein Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from 293 and PANC-1 cells using RagA (D8B5) Rabbit mAb.
    Rag Protein Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from various cell lines using RagD Antibody.
    Rag Protein Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from various cell lines using RagC (D31G9) XP® Rabbit mAb.
    Rag Protein Antibody Sampler Kit: Image 8 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.
    Rag Protein Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from various cell lines using RagB (D18F3) Rabbit mAb.
    Rag Protein Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-) or SignalSilence® mTOR siRNA II (+), using mTOR (7C10) Rabbit mAb #2983 and α-Tubulin (11H10) Rabbit mAb #2125. mTOR (7C10) Rabbit mAb confirms silencing of mTOR expression, while the α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of mTOR siRNA.
    Rag Protein Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis of extracts from mIMCD3, KNRK and COS7 cells using RagA (D8B5) Rabbit mAb.
    Rag Protein Antibody Sampler Kit: Image 12 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse colon using RagC (D31G9) XP® Rabbit mAb.
    Rag Protein Antibody Sampler Kit: Image 13 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma, showing cytoplasmic localization using mTOR (7C10) Rabbit mAb.
    Rag Protein Antibody Sampler Kit: Image 14 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma using RagC (D31G9) XP® Rabbit mAb.
    Rag Protein Antibody Sampler Kit: Image 15 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using mTOR (7C10) Rabbit mAb in the presence of control peptide (left) or mTOR Blocking Peptide #1072 (right).
    Rag Protein Antibody Sampler Kit: Image 16 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lymphoma using RagC (D31G9) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
    Rag Protein Antibody Sampler Kit: Image 17 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse brain using mTOR (7C10) Rabbit mAb.
    Rag Protein Antibody Sampler Kit: Image 18 Expand Image
    Confocal immunofluorescent analysis of mouse embryonic fibroblast (MEF) cells using mTOR (7C10) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Rag Protein Antibody Sampler Kit: Image 19 Expand Image
    Flow cytometric analysis of A549 cells using mTOR (7C10) 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.