Cell Signaling Technology

Product Pathways - Autophagy Signaling

Autophagy Vesicle Elongation (Atg12 Conjugation) Antibody Sampler Kit #32345

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REACTIVITY

No. Size Price
32345T 1 Kit ( 4 x 20 µl ) ¥3,986.00 现货查询 购买询价 防伪查询
Kit Includes Quantity Applications Reactivity Homology† MW (kDa) Isotype
Atg5 (D5F5U) Rabbit mAb #12994 20 µl W,IP, H,M,R, Mk,X,Dg,Pg, 55 Rabbit IgG
Atg12 (D88H11) Rabbit mAb #4180 20 µl W,IP, H,M,R,Mk, 16, 55 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl W, Goat
Atg16L1 (D6D5) Rabbit mAb #8089 20 µl W,IP,IF-IC, H,M,R, Mk, 66, 68 Rabbit IgG
Atg7 (D12B11) Rabbit mAb #8558 20 µl W,IP, H,M,R, Mk,B, 78 Rabbit IgG

Specificity / Sensitivity

Atg5 (D5F5U) Rabbit mAb recognizes endogenous levels of total Atg5 protein. This antibody is capable of detecting Atg5 conjugated to Atg12. Atg12 (D88H11) Rabbit mAb detects endogenous levels of total free and Atg5 bound Atg12 protein. Atg16L1 (D6D5) Rabbit mAb recognizes endogenous levels of total Atg16L1 protein. A background band is detected at 40 kDa in some cell lines. Atg7 (D12B11) Rabbit mAb recognizes endogenous levels of total Atg7 protein.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu265 of human Atg5 protein, a synthetic peptide corresponding to residues surrounding Ser36 of human Atg12 protein, a synthetic peptide corresponding to residues near the amino terminus of human Atg16L1 protein, and a synthetic peptide corresponding to residues near the amino terminus of human Atg7 protein.

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.

Western Blotting

Western Blotting

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.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Atg12 (D88H11) Rabbit mAb.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Atg7 (D12B11) Rabbit mAb.

Western Blotting

Western Blotting

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.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Atg16L1 (D6D5) Rabbit mAb.

Western Blotting

Western Blotting

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.

IF-IC

IF-IC

Confocal immunofluorescent analysis of EBSS-starved PANC-1 cells using Atg16L1 (D6D5) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Atg5 (D5F5U) Rabbit mAb.

IP

IP

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.

Western Blotting

Western Blotting

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.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Atg5 (D5F5U) Rabbit mAb #12994.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Atg16L1 (D6D5) Rabbit mAb #8089.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Atg12 (D88H11) Rabbit mAb #4180.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Atg7 (D12B11) Rabbit mAb #8558.

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).

Application References

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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.

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

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

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