Revision 3

#3868Store 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, W-S, IF-IC, FC-FP

REACTIVITY:

H

SENSITIVITY:

Endogenous

MW (kDa):

14, 16

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q9GZQ8

Entrez-Gene Id:

81631

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:10 - 1:50
Immunofluorescence (Immunocytochemistry) 1:1600 - 1:3200
Flow Cytometry (Fixed/Permeabilized) 1:200

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.

Specificity / Sensitivity

LC3B (D11) XP® Rabbit mAb detects endogenous levels of total LC3B protein. Cross-reactivity may occur with other LC3 isoforms. Stronger reactivity is observed with the type II form of LC3B.

Species Reactivity:

Human

Species predicted to react based on 100% sequence homology

Mouse, Rat, Monkey, Bovine, Pig

Source / Purification

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

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 nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting W-S: Simple Western™ 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.
XP is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

限制使用

除非 CST 的合法授书代表以书面形式书行明确同意,否书以下条款适用于 CST、其关书方或分书商提供的书品。 任何书充本条款或与本条款不同的客书条款和条件,除非书 CST 的合法授书代表以书面形式书独接受, 否书均被拒书,并且无效。

专品专有“专供研究使用”的专专或专似的专专声明, 且未专得美国食品和专品管理局或其他外国或国内专管机专专专任何用途的批准、准专或专可。客专不得将任何专品用于任何专断或治专目的, 或以任何不符合专专声明的方式使用专品。CST 专售或专可的专品提供专作专最专用专的客专,且专用于研专用途。将专品用于专断、专防或治专目的, 或专专售(专独或作专专成)或其他商专目的而专专专品,均需要 CST 的专独专可。客专:(a) 不得专独或与其他材料专合向任何第三方出售、专可、 出借、捐专或以其他方式专专或提供任何专品,或使用专品制造任何商专专品,(b) 不得复制、修改、逆向工程、反专专、 反专专专品或以其他方式专专专专专品的基专专专或技专,或使用专品开专任何与 CST 的专品或服专专争的专品或服专, (c) 不得更改或专除专品上的任何商专、商品名称、徽专、专利或版专声明或专专,(d) 只能根据 CST 的专品专售条款和任何适用文档使用专品, (e) 专遵守客专与专品一起使用的任何第三方专品或服专的任何专可、服专条款或专似专专

Revision 3
#3868

LC3B (D11) XP® Rabbit mAb

Western Blotting Image 1: LC3B (D11) XP® Rabbit mAb Expand Image
使用 LC3B (D11) XP® Rabbit mAb #3868(上图)或 GAPDH (D16H11) XP® Rabbit mAb #5174(下图)对未经处理 (-) 或经过 Chloroquine #14774(50 μM,18 小时)处理的 HCT 116 和 HCT 116 LC3B 敲除型细胞的提取物进行蛋白质印迹分析。HCT 116 敲除型细胞中没有信号,这证实了抗体对 LC3B 的特异性。
Western Blotting Image 2: LC3B (D11) XP® Rabbit mAb Expand Image
使用 LC3B (D11) XP® Rabbit mAb #3868 and α-Tubulin (11H10) Rabbit mAb #2125 对用 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-)、SignalSilence® LC3B siRNA I #6212 (+) 或 SignalSilence® LC3B siRNA II #6213 (+) 转染的 HeLa 细胞提取物进行蛋白质印迹分析。LC3B (D11) XP® Rabbit mAb 可确认 LC3B 的表达沉默,而 α-Tubulin (11H10) Rabbit mAb 则用于 LC3B siRNA 的上样和特异性对照。
Western Blotting Image 1: LC3B (D11) XP® Rabbit mAb Expand Image

使用 LC3B (D11) XP® Rabbit mAb #3868 对来自氯喹(50uM,O/N)处理的 HeLa 细胞的裂解物 (1mg/ml) 进行 Simple Western™ 分析。虚拟泳道式图像(左图)显示一抗稀释比例为 1:10 和 1:50 时的靶标条带(如图所示)。对应的电泳图(右图)为一抗稀释比例在 1:10(蓝线)和 1:50(绿线)时沿毛细血管内分子量的化学发光结果。使用 2-40 kDa 分离模块在 BioTechne 旗下品牌 ProteinSimple 的 Jess™ Simple Western 仪上还原性条件下进行该实验。

Immunofluorescence Image 1: LC3B (D11) XP® Rabbit mAb Expand Image
使用 LC3B (D11) XP® Rabbit mAb(绿色)对未经处理(左图)或经过 Chloroquine #14774(50 µM,过夜)(中图)处理的 HCT 116 细胞或经过 Chloroquine #14774(50 µM,过夜)(右图)处理的 LC3B HCT 116 敲除型细胞进行共聚焦免疫荧光分析。肌动蛋白丝用 β-Actin (8H10D10) Mouse mAb(红色)标记,胞核用 DAPI #4083(蓝色)标记。
Flow Cytometry Image 1: LC3B (D11) XP® Rabbit mAb Expand Image
使用 LC3B (D11) XP® Rabbit mAb #3868(实线)或浓度匹配的 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(虚线)对野生型(绿色,高表达)或 LC3B 敲除型(蓝色,低表达)的 HCT-116 细胞进行共聚焦免疫荧光分析。Anti-rabbit IgG (H+L)、F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 作为二抗。