Revision 13

#83506Store at -20C

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.
Applications:

WB, IP, IHC-P, IF-F, IF-IC, FC-FP

REACTIVITY:

H M R

SENSITIVITY:

Endogenous

MW (kDa):

14, 16

Source/Isotype:

Mouse IgG2b

UniProt ID:

#Q9GZQ8

Entrez-Gene Id:

81631

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:100
Immunohistochemistry (Paraffin) 1:50 - 1:200
Immunofluorescence (Frozen) 1:50 - 1:200
Immunofluorescence (Immunocytochemistry) 1:200 - 1:800
Flow Cytometry (Fixed/Permeabilized) 1:100 - 1:400

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.

For a carrier free (BSA and azide free) version of this product see product #20371.

Specificity / Sensitivity

LC3B (E5Q2K) Mouse mAb recognizes endogenous levels of total LC3B protein. This antibody detects both type I and type II forms of LC3B. Cross reactivity was not detected with other family members.

Species Reactivity:

Human, Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human LC3B protein.

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 posttranslational modifications during autophagy (6-9). 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 (6-10). 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 (11).

  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, 11492-97.
  5. Lang, T. et al. (1998) EMBO J. 17, 3597-607.
  6. Kabeya, Y. et al. (2000) EMBO J. 19, 5720-28.
  7. He, H. et al. (2003) J. Biol. Chem. 278, 29278-87.
  8. Tanida, I. et al. (2004) J. Biol. Chem. 279, 47704-10.
  9. Wu, J. et al. (2006) Biochem. Biophys. Res. Commun. 339, 437-42.
  10. Ichimura, Y. et al. (2000) Nature 408, 488-92.
  11. Kabeya, Y. et al. (2004) J. Cell Sci. 117, 2805-12.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation IHC-P: Immunohistochemistry (Paraffin) IF-F: Immunofluorescence (Frozen) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized)

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
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.

Revision 13
#83506

LC3B (E5Q2K) Mouse mAb

Western Blotting Image 1: LC3B (E5Q2K) Mouse mAb Expand Image
Western blot analysis of extracts from HCT 116 and HCT 116 LC3B knockout cells, untreated (-) or treated with Chloroquine #14774 (50 μM, 18 hr) using LC3B (E5Q2K) Mouse mAb #83506 (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The absence of signal in the HCT 116 knockout cells confirms the specificity of the antibody for LC3B.
Western Blotting Image 2: LC3B (E5Q2K) Mouse mAb Expand Image
Western blot analysis of extracts from HeLa, C2C12, and KNRK cells, untreated (-) or treated with Chloroquine (50 μM, overnight; +) #14774 using LC3B (E5Q2K) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western Blotting Image 3: LC3B (E5Q2K) Mouse mAb Expand Image
Western blot analysis of extracts from HeLa cells or HeLa cells with a knockout of LC3B (HeLa/LC3B KO) using LC3B (E5Q2K) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western Blotting Image 4: LC3B (E5Q2K) Mouse mAb Expand Image
Western blot analysis of extracts from A549 cells, untreated (-) or starved with Earle's Balanced Salt Solution (EBSS) (indicated times) using LC3B (E5Q2K) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb (lower).
Western Blotting Image 5: LC3B (E5Q2K) Mouse mAb Expand Image
Western blot analysis of extracts from MCF7 and Raji cells, untreated (-) or treated with Torin 1 (250 nM, 5 hr; +) #14379, using LC3B (E5Q2K) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Immunoprecipitation Image 1: LC3B (E5Q2K) Mouse mAb Expand Image
Immunoprecipitation of LC3B from HeLa cells treated with Chloroquine (50 μM, overnight) # 14774. Lane 1 is 10% input, lane 2 is Mouse (G3A1) mAb IgG1 Isotype Control, and lane 3 is LC3B (E5Q2K) Mouse mAb. Western blot was performed using LC3B (E5Q2K) Mouse mAb. Anti-mouse IgG, HRP-linked Antibody #7076 was used as a secondary antibody.
Immunohistochemistry Image 1: LC3B (E5Q2K) Mouse mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using LC3B (E5Q2K) Mouse mAb.
Immunohistochemistry Image 2: LC3B (E5Q2K) Mouse mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human esophageal carcinoma using LC3B (E5Q2K) Mouse mAb.
Immunohistochemistry Image 3: LC3B (E5Q2K) Mouse mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human lung carcinoma using LC3B (E5Q2K) Mouse mAb.
Immunohistochemistry Image 4: LC3B (E5Q2K) Mouse mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human non-Hodgkin's lymphoma using LC3B (E5Q2K) Mouse mAb.
Immunohistochemistry Image 5: LC3B (E5Q2K) Mouse mAb Expand Image
Immunohistochemical analysis of paraffin-embedded HCT116 cell pellets, untreated (left-top) or treated with Chloroquine #14774 (right-top), and HCT116 LC3B knockout cell pellets, untreated (left-bottom) or treated with Chloroquine #14774 (right-bottom), using LC3B (E5Q2K) Mouse mAb.
Immunohistochemistry Image 6: LC3B (E5Q2K) Mouse mAb Expand Image
Immunohistochemical analysis of paraffin-embedded normal human brain using LC3B (E5Q2K) Mouse mAb.
Immunohistochemistry Image 7: LC3B (E5Q2K) Mouse mAb Expand Image
Immunohistochemical analysis of paraffin-embedded KARPAS 299 cell pellet (left, high-expressing) or VCaP cell pellet (right, low-expressing) using LC3B (E5Q2K) Mouse mAb.
Immunohistochemistry Image 8: LC3B (E5Q2K) Mouse mAb Expand Image
Immunohistochemical analysis of paraffin-embedded HeLa cell pellets, untreated (left) or treated with Chloroquine #14774 (right), using LC3B (E5Q2K) Mouse mAb.
Immunohistochemistry Image 9: LC3B (E5Q2K) Mouse mAb Expand Image
Immunohistochemical analysis of paraffin-embedded normal human spleen using LC3B (E5Q2K) Mouse mAb.
Immunofluorescence Image 1: LC3B (E5Q2K) Mouse mAb Expand Image
Confocal immunofluorescent analysis of fixed frozen mouse cerebellum, labeled with LC3B (E5Q2K) Mouse mAb #83506 (left, green) and co-labeled with GFAP (E4L7M) XP® Rabbit mAb #80788 (right, red) and DAPI #4083 (right, blue).
Immunofluorescence Image 2: LC3B (E5Q2K) Mouse mAb Expand Image
Confocal immunofluorescent analysis of fixed frozen mouse pons, labeled with LC3B (E5Q2K) Mouse mAb #83506 (left, green) and co-labeled with GFAP (E4L7M) XP® Rabbit mAb #80788 (right, red) and DAPI #4083 (right, blue).
Immunofluorescence Image 1: LC3B (E5Q2K) Mouse mAb Expand Image
Confocal immunofluorescent analysis of HCT 116 cells either untreated (left) or treated with Chloroquine #14774 (50 µM, overnight) (center) or LC3B HCT 116 knockout cells treated with Chloroquine #14774 (50 µM, overnight) (right) using LC3B (E5Q2K) Mouse mAb (green). Actin filaments were labeled with β-Actin (13E5) Rabbit mAb #4970 (red) and nuclei were labeled with DAPI #4083 (blue).
Flow Cytometry Image 1: LC3B (E5Q2K) Mouse mAb Expand Image
Flow cytometric analysis of HCT-116 cells, untreated (blue) or treated with Chloroquine #14774 (50 μM, 18 hr; green) using LC3B (E5Q2K) Mouse mAb Antibody (solid lines) or concentration-matched Mouse (E7Q5L) mAb IgG2b Isotype Control #53484 (dashed lines). Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.
Flow Cytometry Image 2: LC3B (E5Q2K) Mouse mAb Expand Image
Flow cytometric analysis of HCT-116 cells, wild-type (green, high expression) or LC3B knockdown (blue, negative expression), using LC3B (E5Q2K) Mouse mAb or a concentration-matched Mouse (E7Q5L) mAb IgG2b Isotype Control #53484 (dashed lines). Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.