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19848
Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit
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Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit #19848

Citations (1)

Simple Western™ analysis of lysates (0.1 mg/mL) from 3T3 cells using LC3A/B (D3U4C) XP® Rabbit mAb #12741. 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 2 - 40 kDa separation module.

Western blot analysis of extracts from RD cells, untreated (-) or Torin 1-treated (250 nM, 4 hr; +), using LC3A/B (D3U4C) XP® Rabbit mAb.
Western blot analysis of extracts from various cell lines using Atg4B (D1G2R) Rabbit mAb.
Western blot analysis of A172 and C2C12 cells, untreated (-) or chloroquine-treated (50 μM, overnight; +), using GABARAP (E1J4E) Rabbit mAb.
Western blot analysis of extracts from various cell lines using Atg3 Antibody.
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 blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® Atg4A siRNA I #6427 (+), using Atg4A (D62C10) Rabbit mAb (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The Atg4A (D62C10) Rabbit mAb confirms silencing of Atg4A expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.
Western blot analysis of extracts from various cell lines using Atg7 (D12B11) Rabbit mAb.
Western blot analysis of extracts from HeLa, NIH/3T3, and KNRK cells, untreated (-) or chloroquine-treated (50 μM, overnight; +), using LC3A/B (D3U4C) XP® Rabbit mAb.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing full-length mouse Atg4B (mAtg4B; +), using Atg4B (D1G2R) Rabbit mAb.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human GABARAP (hGABARAP-Myc/DDK; +), human GABARAPL1 (hGABARAPL1-Myc/DDK; +), or human GABARAPL2 (hGABARAPL2-Myc/DDK; +), using GABARAP (E1J4E) Rabbit mAb.
Western blot analysis of extracts from HeLa cells, mock transfected or transfected with mouse Atg3, using Atg3 Antibody.
Western blot analysis of extracts from various cell lines using Atg4A (D62C10) Rabbit mAb.
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.
Immunohistochemical analysis of paraffin-embedded mouse prostate using LC3A/B (D3U4C) XP® Rabbit mAb.
Western blot analysis of extracts from RD cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® Atg4B siRNA I #6336 (+), using Atg4B (D1G2R) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The Atg4B (D1G2R) Rabbit mAb confirms silencing of Atg4B expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.
Confocal immunofluorescent analysis of A172 cells, untreated (left) and chloroquine-treated (50 μM, overnight; right), using GABARAP (E1J4E) Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a DYKDDDDK-tagged human Atg4A construct (+), using Atg4A (D62C10) Rabbit mAb (upper) and DYKDDDDK Tag Antibody #2368 (lower).
Immunohistochemical analysis of paraffin-embedded human squamous cell lung carcinoma using LC3A/B (D3U4C) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
Immunoprecipitation of Atg4B from RD cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype control #3900 (lane 2) or Atg4B (D1G2R) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Atg4B (D1G2R) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded NIH/3T3 cell pellets, control (left) or chloroquine-treated (right), using LC3A/B (D3U4C) XP® Rabbit mAb.
Confocal immunofluorescent analysis of HeLa (upper) and C2C12 (lower) cells, chloroquine-treated (50 μM, overnight; left), nutrient-starved with EBSS (3 hr, middle) or untreated (right) using LC3A/B (D3U4C) XP® Rabbit mAb (green) and β-Actin (13E5) Rabbit mAb (Alexa Fluor® 555 Conjugate) #8046 (red). Blue pseudocolor= DRAQ5® #4084 (fluorescent DNA dye).
Flow cytometric analysis of HeLa cells, untreated (blue) or treated with chloroquine (50 µM, 16 hr; green) using LC3A/B (D3U4C) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.
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To Purchase # 19848
Cat. # Size Qty. Price Inventory
19848T		 1 Kit  (6 x 20 microliters)

Custom Formulation

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
LC3A/B (D3U4C) XP® Rabbit mAb 12741 20 µl
  • WB
  • IHC
  • IF
  • F
 H M R 14, 16 Rabbit IgG
Atg7 (D12B11) Rabbit mAb 8558 20 µl
  • WB
  • IP
 H M R 78 Rabbit IgG
Atg4B (D1G2R) Rabbit mAb 13507 20 µl
  • WB
  • IP
 H M R 48 Rabbit IgG
Atg4A (D62C10) Rabbit mAb 7613 20 µl
  • WB
  • IP
 H R 48-60 Rabbit IgG
GABARAP (E1J4E) Rabbit mAb 13733 20 µl
  • WB
  • IF
 H M R 14, 16 Rabbit IgG
Atg3 Antibody 3415 20 µl
  • WB
 H M R Mk 40 Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
 Rab Goat 

Product Description

The Autophagy Vesicle Elongation (LC3 Conjugation) Antibody Sampler Kit provides an economical means of detecting target proteins related to autophagy vesicle elongation pathway. The kit contains enough antibody to perform two western blots per primary.

Specificity / Sensitivity

LC3A/B (D3U4C) XP® Rabbit mAb recognizes endogenous levels of total LC3A and LC3B proteins. Atg7 (D12B11) Rabbit mAb recognizes endogenous levels of total Atg7 protein. Atg4B (D1G2R) Rabbit mAb recognizes endogenous levels of total Atg4B protein. Atg4A (D62C10) Rabbit mAb recognizes endogenous levels of total Atg4A protein and recognizes unidentified bands within the molecular weight range of 48-60 kDa, which decrease with silencing of Atg4A expression. GABARAP (E1J4E) Rabbit mAb recognizes endogenous levels of total GABARAP protein, but does not cross-react with other GABARAP family members. Atg3 Antibody detects endogenous levels of total Atg3 protein.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu44 of human LC3B protein (conserved in LC3A), a synthetic peptide corresponding to residues near the amino terminus of human Atg7 protein, a synthetic peptide corresponding to residues surrounding Gln100 of human Atg4B protein, a synthetic peptide corresponding to residues near the carboxy terminus of human Atg4A protein, a synthetic peptide corresponding to residues surrounding Arg40 of human GABARAP protein. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of Atg3. Antibodies are purified by protein A and peptide affinity chromatography.

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 it has also been associated with a number of physiological processes including development, differentiation, neurodegenerative diseases, infection, and cancer (3). Autophagy marker Light Chain 3 (LC3) was originally identified as a subunit of microtubule-associated proteins 1A and 1B (termed MAP1LC3) (4) and subsequently found to contain similarity to the yeast protein Apg8/Aut7/Cvt5 critical for autophagy (5). Three human LC3 isoforms (LC3A, LC3B, and LC3C) undergo post-translational modifications during autophagy (6-8). Cleavage of LC3 at the carboxy terminus immediately following synthesis yields the cytosolic LC3-I form. During autophagy, LC3-I is converted to LC3-II through lipidation by a ubiquitin-like system involving Atg7 and Atg3 that allows for LC3 to become associated with autophagic vesicles (6-9). The presence of LC3 in autophagosomes and the conversion of LC3 to the lower migrating form, LC3-II, have been used as indicators of autophagy (10). Numerous mammalian counterparts to yeast Atg proteins have been described, including three Atg8 proteins (GATE-16, GABARAP, and LC3) and four Atg4 homologs (Atg4A/autophagin-2, Atg4B/autophagin-1, Atg4C/autophagin-3, and Atg4D/autophagin-4) (10-12). The cysteine protease Atg4 is pivotal to autophagosome membrane generation and regulation (13). GABAA receptor associated protein (GABARAP) is an Atg8 family protein with a key role in autophagy, which was originally discovered as a protein associated with the GABAA receptor regulating receptor trafficking to the plasma membrane (14). Processing of GABARAP involves cleavage by Atg4 family members (15,16) followed by conjugation by the E1 and E2 like enzymes Atg7 and Atg3 (17,18).

  1. Reggiori, F. and Klionsky, D.J. (2002) Eukaryot Cell 1, 11-21.
  2. Codogno, P. and Meijer, A.J. (2005) Cell Death Differ 12 Suppl 2, 1509-18.
  3. Levine, B. and Yuan, J. (2005) J Clin Invest 115, 2679-88.
  4. Mann, S.S. and Hammarback, J.A. (1994) J Biol Chem 269, 11492-7.
  5. Lang, T. et al. (1998) EMBO J 17, 3597-607.
  6. He, H. et al. (2003) J Biol Chem 278, 29278-87.
  7. Tanida, I. et al. (2004) J Biol Chem 279, 47704-10.
  8. Wu, J. et al. (2006) Biochem Biophys Res Commun 339, 437-42.
  9. Ichimura, Y. et al. (2000) Nature 408, 488-92.
  10. Kabeya, Y. et al. (2004) J Cell Sci 117, 2805-12.
  11. Kabeya, Y. et al. (2000) EMBO J 19, 5720-8.
  12. Mariño, G. et al. (2003) J Biol Chem 278, 3671-8.
  13. Sou, Y.S. et al. (2008) Mol Biol Cell 19, 4762-75.
  14. Wang, H. et al. (1999) Nature 397, 69-72.
  15. Tanida, I. et al. (2004) J Biol Chem 279, 36268-76.
  16. Hemelaar, J. et al. (2003) J Biol Chem 278, 51841-50.
  17. Tanida, I. et al. (2001) J Biol Chem 276, 1701-6.
  18. Tanida, I. et al. (2002) J Biol Chem 277, 13739-44.

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