Revision 1

#64459Store at -20C

1 Kit

(6 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

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For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
LC3A (D50G8) XP® Rabbit mAb 4599 20 µl 14, 16 kDa Rabbit IgG
LC3B (D11) XP® Rabbit mAb 3868 20 µl 14, 16 kDa Rabbit IgG
LC3C (D3O6P) Rabbit mAb 14736 20 µl 14 kDa Rabbit IgG
GABARAP (E1J4E) Rabbit mAb 13733 20 µl 14, 16 kDa Rabbit IgG
GABARAPL1 (D5R9Y) XP® Rabbit mAb 26632 20 µl 14, 16 kDa Rabbit IgG
GABARAPL2 (D1W9T) Rabbit mAb 14256 20 µl 14 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

The Autophagy Atg8 Family Antibody Sampler Kit provides an economical means of detecting each of the Atg8 family members. The kit contains enough primary antibody to perform at least two western blot experiments.

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.

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).
Atg8 is a ubiquitin-like protein that is critical for autophagosome formation. Atg8 is synthesized as a precursor protein that is processed by the cysteine protease Atg4, followed by lipidation with phosphatidylethanolamine (PE) in a ubiqutin-like conjugation pathway involving Atg7 and Atg3 (4). This processing of Atg8, which is described as a conversion from type-I to type-II forms, is frequently described as a marker for autophagy. The type-II form of Atg8 is incorporated into maturing autophagosomes and leads to the recruitment of additional autophagy components, including cargo receptors like SQSTM1/p62. While yeast has a single Atg8 gene, many eukaryotes have at least six orthologs, including three microtubule-associated protein 1 light chain 3 (MAP1LC3/LC3) family members (LC3A, LC3B, and LC3C) and three GABAA receptor associated protein (GABARAP) family members (GABARAP, GABARAPL1/GEC1, and GABARAPL2/GATE-16). While highly conserved, these various family members can have important differences in their post-translational processing, expression profile, and protein interactions including distinct cargo receptor. This complexity within the Atg8 family is critical for selective mechanisms of autophagy that have been reported (5, 6).

  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. Ichimura, Y. et al. (2000) Nature 408, 488-92.
  5. Slobodkin, M.R. and Elazar, Z. (2013) Essays Biochem 55, 51-64.
  6. Schaaf, M.B. et al. (2016) FASEB J 30, 3961-3978.

Background References

    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.

    使用に関する制限

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    研究専用 (For Research Use Only) またはこれに類似する表示がされた製品は、 いかなる目的についても FDA または外国もしくは国内のその他の規制機関により承認、認可または許可を受けていません。 お客様は製品を診断もしくは治療目的で使用してはならず、また、製品に表示された内容に違反する方法で使用してはなりません。 CST が販売または使用許諾する製品は、エンドユーザーであるお客様に対し、使途を研究および開発のみに限定して提供されるものです。 診断、予防もしくは治療目的で製品を使用することまたは製品を再販売 (単独であるか他の製品等の一部であるかを問いません) もしくはその他の商業的利用の目的で購入することについては、CST から別途許諾を得る必要があります。 お客様は以下の事項を遵守しなければなりません。(a) CST の製品 (単独であるか他の資材と一緒であるかを問いません) を販売、使用許諾、貸与、寄付もしくはその他の態様で第三者に譲渡したり使用させたりしてはなりません。また、商用の製品を製造するために CST の製品を使用してはなりません。(b) 複製、改変、リバースエンジニアリング、逆コンパイル、 分解または他の方法により製品の構造または技術を解明しようとしてはなりません。また、 CST の製品またはサービスと競合する製品またはサービスを開発する目的で CST の製品を使用してはなりません。(c) CST の製品の商標、商号、ロゴ、特許または著作権に関する通知または表示を除去したり改変したりしてはなりません。(d) CST の製品をCST 製品販売条件(CST’s Product Terms of Sale) および該当する書面のみに従って使用しなければなりません。(e) CST の製品に関連してお客様が使用する第三者の製品またはサービスに関する使用許諾条件、 サービス提供条件またはこれに類する合意事項を遵守しなければなりません。

    Revision 1
    #64459

    Autophagy Atg8 Family Antibody Sampler Kit

    Autophagy Atg8 Family Antibody Sampler Kit: Image 1 Expand Image

    Simple Western™ analysis of lysates (1mg/ml) from HeLa cells treated with Chloroquine (50uM, O/N) using LC3B (D11) XP® Rabbit mAb #3868. 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 2-40kDa.

    Autophagy Atg8 Family Antibody Sampler Kit: Image 2 Expand Image
    Western blot analysis of A172 and C2C12 cells, untreated (-) or chloroquine-treated (50 μM, overnight; +), using GABARAP (E1J4E) Rabbit mAb.
    Autophagy Atg8 Family Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from 293T cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® GABARAPL2 siRNA I # 14239 (+) or SignalSilence® II GABARAPL2 siRNA II #14246 (+), using GABARAPL2 (D1W9T) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The GABARAPL2 (D1W9T) Rabbit mAb confirms silencing of GABARAPL2, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.
    Autophagy Atg8 Family Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from A172 and HOP-92 cells, untreated (-) or chloroquine-treated (50 μM, overnight; +), using LC3C (D3O6P) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Autophagy Atg8 Family Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human GABARAP protein (hGABARAP-Myc/DDK; +), human GABARAPL1 protein (hGABARAPL1-Myc/DDK; +), or human GABARAPL2 protein (hGABARAPL2-Myc/DDK; +) using GABARAPL1 (D5R9Y) XP® Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Autophagy Atg8 Family Antibody Sampler Kit: Image 6 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 (D11) XP® Rabbit mAb #3868 (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.
    Autophagy Atg8 Family Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from HeLa and NIH/3T3 cells, untreated or chloroquine-treated (50 μM, overnight), using LC3A (D50G8) XP® Rabbit mAb.
    Autophagy Atg8 Family Antibody Sampler Kit: Image 8 Expand Image
    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.
    Autophagy Atg8 Family Antibody Sampler Kit: Image 9 Expand Image
    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.
    Autophagy Atg8 Family Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from various cell lines using GABARAPL2 (D1W9T) Rabbit mAb.
    Autophagy Atg8 Family Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing full-length human LC3A protein (hLC3A; +), human LC3B protein (hLC3B; +), or human LC3C protein (hLC3C; +), using LC3C (D3O6P) Rabbit mAb.
    Autophagy Atg8 Family Antibody Sampler Kit: Image 12 Expand Image
    Western blot analysis of extracts from HeLa, C2C12, and C6 cells, untreated (-) or treated with chloroquine (50 μM, overnight; +), using GABARAPL1 (D5R9Y) XP® Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Autophagy Atg8 Family Antibody Sampler Kit: Image 13 Expand Image
    Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® LC3B siRNA I #6212 (+) or SignalSilence® LC3B siRNA II #6213 (+), using LC3B (D11) XP® Rabbit mAb #3868 and α-Tubulin (11H10) Rabbit mAb #2125. The LC3B (D11) XP® Rabbit mAb confirms silencing of LC3B expression, while the α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of LC3B siRNA.
    Autophagy Atg8 Family Antibody Sampler Kit: Image 14 Expand Image
    Immunohistochemical analysis of paraffin-embedded human glioblastoma multiforme using LC3A (D50G8) XP® Rabbit mAb.
    Autophagy Atg8 Family Antibody Sampler Kit: Image 15 Expand Image
    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).
    Autophagy Atg8 Family Antibody Sampler Kit: Image 16 Expand Image
    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 GABARAPL2 (D1W9T) Rabbit mAb.
    Autophagy Atg8 Family Antibody Sampler Kit: Image 17 Expand Image
    Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with chloroquine (50 μM, 24 hr; right), using GABARAPL1 (D5R9Y) XP® Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Autophagy Atg8 Family Antibody Sampler Kit: Image 18 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma using LC3A (D50G8) XP® Rabbit mAb (left) or

    Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).

    Autophagy Atg8 Family Antibody Sampler Kit: Image 19 Expand Image
    Western blot analysis of extracts from A172 and HeLa cells, untreated (-) or treated overnight with chloroquine (50 μM) (+), using GABARAPL2 (D1W9T) Rabbit mAb (upper) or β-Actin (D6A8) #8457 (lower).
    Autophagy Atg8 Family Antibody Sampler Kit: Image 20 Expand Image
    Flow cytometric analysis of HeLa cells using GABARAPL1 (D5R9Y) XP® Rabbit mAb (blue) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    Autophagy Atg8 Family Antibody Sampler Kit: Image 21 Expand Image
    Immunohistochemical analysis of paraffin-embedded HeLa cell pellets, control (left) or choloroquine-treated (right), using LC3A (D50G8) XP® Rabbit mAb.
    Autophagy Atg8 Family Antibody Sampler Kit: Image 22 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 (D11) XP® Rabbit mAb (green). Actin filaments were labeled with β-Actin (8H10D10) Mouse mAb (red) and nuclei were labeled with DAPI #4083 (blue).
    Autophagy Atg8 Family Antibody Sampler Kit: Image 23 Expand Image
    Flow cytometric analysis of HCT-116 cells, wild-type (green, high expression) or LC3B knockdown (blue, negative expression), using LC3B (D11) XP® Rabbit mAb #3868 (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.