Revision 2

#32345

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Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Product Includes Product # Quantity Mol. Wt Isotype/Source
Atg5 (D5F5U) Rabbit Monoclonal Antibody1299420 µl55 kDaRabbit IgG
Atg12 (D88H11) Rabbit Monoclonal Antibody418020 µl16, 55 kDaRabbit IgG
Atg16L1 (D6D5) Rabbit Monoclonal Antibody808920 µl66, 68 kDaRabbit IgG
Atg7 (D12B11) Rabbit Monoclonal Antibody855820 µl78 kDaRabbit IgG
Anti-rabbit IgG, HRP-linked Antibody7074100 µlGoat

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

Description

The Autophagy Vesicle Elongation (Atg12 Conjugation) Antibody Sampler Kit provides an economical means of detecting proteins related to autophagy vesicle elongation pathway. The kit contains enough antibody to perform two 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 antibodies.

Background

Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic contents (1,2). Autophagy is generally activated by conditions of nutrient deprivation but has also been associated with a number of physiological processes including development, differentiation, neurodegeneration, infection, and cancer (3). The molecular machinery of autophagy was largely discovered in yeast and referred to as autophagy-related (Atg) genes. Formation of the autophagosome involves a ubiquitin-like conjugation system in which Atg12 is covalently bound to Atg5 and targeted to autophagosome vesicles (4-6). This conjugation reaction is mediated by the ubiquitin E1-like enzyme Atg7 and the E2-like enzyme Atg10 (7,8). Atg16L1 binds Atg5 of the Atg12-Atg5 conjugate forming an 800 kDa multimeric complex (9). The Atg12-Atg-5-Atg16L1 complex localizes to pre-autophagosomal membranes where it determines the site of LC3 lipidation and catalyzes the reaction required for the formation of mature autophagosomes (9,10).

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

KARPAS cell line source: Dr. Abraham Karpas at the University of Cambridge.

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.

Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
Western blot analysis of extracts from various cell lines using Atg5 (D5F5U) Rabbit mAb.
undefined Image 1: Autophagy Vesicle Elongation (Atg12 Conjugation) Antibody Sampler Kit
Western blot analysis of extracts from various cell lines using Atg12 (D88H11) Rabbit mAb.
undefined Image 2: Autophagy Vesicle Elongation (Atg12 Conjugation) Antibody Sampler Kit
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.
undefined Image 3: Autophagy Vesicle Elongation (Atg12 Conjugation) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
Western blot analysis of extracts from various cell lines using Atg16L1 (D6D5) Rabbit mAb.
undefined Image 4: Autophagy Vesicle Elongation (Atg12 Conjugation) Antibody Sampler Kit
Western blot analysis of extracts from various cell lines using Atg7 (D12B11) Rabbit mAb.
undefined Image 5: Autophagy Vesicle Elongation (Atg12 Conjugation) Antibody Sampler Kit
Western blot analysis of extracts from wild-type MEFs (wt) or MEFs from Atg5 knockouts (Atg5-/-) using Atg5 (D5F5U) Rabbit mAb (upper), or β-Actin (D6A8) Rabbit mAb #8457 (lower). Atg5-/- MEFs were kindly provided by Dr. Ramnik Xavier, Massachusetts General Hospital, Harvard Medical School, Boston, MA.
undefined Image 6: Autophagy Vesicle Elongation (Atg12 Conjugation) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with either a myc-tagged human Atg16L1β construct (hAtg16L1β-Myc; +) or a mouse Atg16L1α construct (mAtg16L1α; +), using Atg16L1 (D6D5) Rabbit mAb. The myc-tagged human Atg16L1β construct was kindly provided by Dr. Qing Zhong, University of California Berkeley.
undefined Image 7: Autophagy Vesicle Elongation (Atg12 Conjugation) Antibody Sampler Kit
Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® Atg7 siRNA I #6604 (+), using Atg7 (D12B11) Rabbit mAb (upper) or α-Tubulin (11Η10) Rabbit mAb #2125 (lower). The Atg7 (D12B11) Rabbit mAb confirms silencing of Atg7 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.
undefined Image 8: Autophagy Vesicle Elongation (Atg12 Conjugation) Antibody Sampler Kit
Immunoprecipitation of Atg5 from PANC-1 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Atg5 (D5F5U) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot was performed using Atg5 (D5F5U) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 was used as a secondary antibody to avoid cross-reactivity with rabbit IgG.
undefined Image 9: Autophagy Vesicle Elongation (Atg12 Conjugation) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
Confocal immunofluorescent analysis of EBSS-starved PANC-1 cells using Atg16L1 (D6D5) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
undefined Image 10: Autophagy Vesicle Elongation (Atg12 Conjugation) Antibody Sampler Kit
Simple WesternTM analysis of lysates (0.1 mg/mL) from HeLa cells using Atg5 (D5F5U) Rabbit mAb #12994. The virtual lane view (left) shows the target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the JessTM Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
undefined Image 11: Autophagy Vesicle Elongation (Atg12 Conjugation) Antibody Sampler Kit
Immunoprecipitation of Atg16L1 from Jurkat cell extracts. Lane 1 is 10% input, lane 2 is precipitated with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Atg16L1 (D6D5) Rabbit mAb, #8089. Western blot was performed using Atg16L1 (D6D5) Rabbit mAb.
undefined Image 12: Autophagy Vesicle Elongation (Atg12 Conjugation) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
Simple Western™ analysis of lysates (0.1 mg/mL) from Ba/F3 cells using Atg7 (D12B11) Rabbit mAb #8558. The virtual lane view (left) shows the 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 12-230 kDa separation module.
undefined Image 13: Autophagy Vesicle Elongation (Atg12 Conjugation) Antibody Sampler Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.