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.

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 13
#83506

LC3B (E5Q2K) Mouse mAb

Western Blotting Image 1: LC3B (E5Q2K) Mouse mAb Expand Image
使用 LC3B (E5Q2K) Mouse mAb #83506(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 (E5Q2K) Mouse mAb Expand Image
使用 LC3B (E5Q2K) 小鼠单克隆抗体(上图)或 β-肌动蛋白 (D6A8) 兔单克隆抗体 #8457(下图)对未经 (-) 或已经氯喹 #14774(50 μM,过夜;+)处理的 HeLa、C2C12 和 KNRK 细胞的提取物进行蛋白印迹分析。
Western Blotting Image 3: LC3B (E5Q2K) Mouse mAb Expand Image
使用 LC3B (E5Q2K) 小鼠单克隆抗体(上图)或 β-肌动蛋白 (D6A8) 兔单克隆抗体 #8457(下图)对 HeLa 细胞或已敲除 LC3B 的 HeLa 细胞 (HeLa/LC3B KO) 的提取物进行蛋白印迹分析。
Western Blotting Image 4: LC3B (E5Q2K) Mouse mAb Expand Image
使用 LC3B (E5Q2K) 小鼠单克隆抗体(上图)或 β-肌动蛋白 (D6A8) 兔单克隆抗体(下图)对未处理的 (-) 或用 Earle's 平衡盐溶液 (EBSS)(规定时间)饥饿的 A549 细胞的提取物进行蛋白印迹分析。
Western Blotting Image 5: LC3B (E5Q2K) Mouse mAb Expand Image
使用 LC3B (E5Q2K) 小鼠单克隆抗体(上图)或 β-肌动蛋白 (D6A8) 兔单克隆抗体 #8457(下图)对未处理的 (-) 或已经 Torin 1 #14379(250 nM,5 小时;+)处理的 MCF7 和 Raji 细胞的提取物进行蛋白印迹分析。
Immunoprecipitation Image 1: LC3B (E5Q2K) Mouse mAb Expand Image
对用氯喹 # 14774(50 μM;过夜)处理的 HeLa 细胞的 LC3B 进行免疫沉淀。泳道 1 为 10% 敲入,泳道 2 为小鼠 (G3A1) 单克隆抗体 IgG1 同型对照,泳道 3 为 LC3B (E5Q2K) 小鼠单克隆抗体。使用 LC3B (E5Q2K) 小鼠单克隆抗体进行蛋白印迹实验。Anti-mouse IgG, HRP-linked Antibody #7076 用作二抗。
Immunohistochemistry Image 1: LC3B (E5Q2K) Mouse mAb Expand Image
使用 LC3B (E5Q2K) 小鼠单克隆抗体对石蜡包埋的人结肠癌细胞进行免疫组织化学分析。
Immunohistochemistry Image 2: LC3B (E5Q2K) Mouse mAb Expand Image
使用 LC3B (E5Q2K) 小鼠单克隆抗体对石蜡包埋的人食管癌细胞进行免疫组织化学分析。
Immunohistochemistry Image 3: LC3B (E5Q2K) Mouse mAb Expand Image
使用 LC3B (E5Q2K) 小鼠单克隆抗体对石蜡包埋的人肺癌细胞进行免疫组织化学分析。
Immunohistochemistry Image 4: LC3B (E5Q2K) Mouse mAb Expand Image
使用 LC3B (E5Q2K) 小鼠单克隆抗体对石蜡包埋的人非霍奇金氏淋巴瘤细胞进行免疫组织化学分析。
Immunohistochemistry Image 5: LC3B (E5Q2K) Mouse mAb Expand Image
使用 LC3B (E5Q2K) Mouse mAb 对未经处理(左上图)或经过 Chloroquine #14774 处理(右上图)的石蜡包埋的 HCT116 细胞沉淀物和未经处理(左下图)或经过 Chloroquine #14774 处理(右下图)的 HCT116 LC3B 敲除型细胞沉淀物进行免疫组织化学分析。
Immunohistochemistry Image 6: LC3B (E5Q2K) Mouse mAb Expand Image
使用 LC3B (E5Q2K) Mouse mAb 对石蜡包埋的正常人脑细胞进行免疫组织化学分析。
Immunohistochemistry Image 7: LC3B (E5Q2K) Mouse mAb Expand Image
使用 LC3B (E5Q2K) Mouse mAb 对石蜡包埋的 KARPAS 299 细胞沉淀物(左图,高表达)或 VCaP 细胞沉淀物(右图,低表达)进行免疫组织化学分析。
Immunohistochemistry Image 8: LC3B (E5Q2K) Mouse mAb Expand Image
使用 LC3B (E5Q2K) Mouse mAb 对石蜡包埋的未处理的(左图)或经过 Chloroquine #14774 处理的(右图)HeLa 细胞沉淀物进行免疫组织化学分析。
Immunohistochemistry Image 9: LC3B (E5Q2K) Mouse mAb Expand Image
使用 LC3B (E5Q2K) Mouse mAb 对石蜡包埋的人正常脾进行免疫组织化学分析。
Immunofluorescence Image 1: LC3B (E5Q2K) Mouse mAb Expand Image
对使用 LC3B (E5Q2K) Mouse mAb #83506(左图,绿色)标记以及使用 GFAP (E4L7M) XP® Rabbit mAb #80788(右图,红色)和 DAPI #4083(右图,蓝色)共同标记的固定冷冻的小鼠小脑组织进行共聚焦免疫荧光分析。
Immunofluorescence Image 2: LC3B (E5Q2K) Mouse mAb Expand Image
对使用 LC3B (E5Q2K) Mouse mAb #83506(左图,绿色)标记以及使用 GFAP (E4L7M) XP® Rabbit mAb #80788(右图,红色)和 DAPI #4083(右图,蓝色)共同标记的固定冷冻的小鼠脑桥组织进行共聚焦免疫荧光分析。
Immunofluorescence Image 1: LC3B (E5Q2K) Mouse mAb Expand Image
使用 LC3B (E5Q2K) Mouse 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 (13E5) Rabbit mAb #4970(红色)标记,胞核用 DAPI #4083(蓝色)标记。
Flow Cytometry Image 1: LC3B (E5Q2K) Mouse mAb Expand Image
使用 LC3B (E5Q2K) Mouse mAb Antibody(实线)或浓度匹配的 Mouse (E7Q5L) mAb IgG2b Isotype Control #53484(虚线)对未经处理(蓝色)或已经过 Chloroquine #14774(50 μM,18 小时;绿色)处理的 HCT-116 细胞进行流式细胞分析。Anti-mouse IgG (H+L),F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 作为二抗。
Flow Cytometry Image 2: LC3B (E5Q2K) Mouse mAb Expand Image
使用 LC3B (E5Q2K) Mouse mAb 或浓度匹配的 Mouse (E7Q5L) mAb IgG2b Isotype Control #53484(虚线)对野生型 HCT-116 细胞(绿色,高表达)或敲除型 LC3B(蓝色,阴性)进行流式细胞分析。Anti-mouse IgG (H+L),F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 作为二抗。