Product datasheet

Anti-LC3B antibody [EPR18709] - Autophagosome Markerab192890
リコンビナント

17 Abreviews 468 References 画像数 12
製品の概要
製品名

Anti-LC3B antibody [EPR18709] - Autophagosome Marker

製品の詳細

Rabbit monoclonal [EPR18709] to LC3B - Autophagosome Marker

由来種

Rabbitアプリケーション

適用あり : IHC-P, WB, ICC/IF
適用なし : IP

種交差性

交差種 : Mouse, Rat, Human

免疫原

Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

ポジティブ･コントロール

WB: BMDM, U-87 MG, C6 and RAW 264.7 whole cell lysates; Human brain, mouse heart, ratheart, mouse brain and rat brain lysates. ICC/IF: HeLa cells (+/- chloroquine), HAP1 cells (+/chloroquine) (HAP1-MAP1LC3B knockout cells used as negative cell line). IHC-P: Human
Cerebral Cortex tissue sections

特記事項

This product is a recombinant monoclonal antibody, which offers several advantages including:- High batch-to-batch consistency and reproducibility- Improved sensitivity and specificity- Long-term security of supply- Animal-free production
For more information see here.
Our RabMAb® technology is a patented hybridoma-based technology for making rabbitmonoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

製品の特性
製品の状態

Liquid

保存方法

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C longterm. Avoid freeze / thaw cycle.

バッファー

pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
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精製度

Protein A purified

ポリ /モノ

モノクローナル

クローン名

EPR18709

アイソタイプ

IgG

アプリケーション

The Abpromise guarantee

Abpromise保証は、 次のテスト済みアプリケーションにおけるab192890の使用に適用されます

アプリケーションノートには、推奨の開始希釈率がありますが、適切な希釈率につきましてはご検討ください。
アプリケーション

特記事項

Abreviews

IHC-P

Use a concentration of 0.1 µg/ml. Perform heat mediated antigen
retrieval with citrate buffer pH 6 before commencing with IHC
staining protocol.

WB

(8)

1/2000. Detects a band of approximately 14, 16 kDa (predicted
molecular weight: 15 kDa).

ICC/IF(6)

Use a concentration of 1 µg/ml.

追加情報

Is unsuitable for IP.

ターゲット情報機能

Probably involved in formation of autophagosomal vacuoles (autophagosomes).

組織特異性

Most abundant in heart, brain, skeletal muscle and testis. Little expression observed in liver.

配列類似性

Belongs to the MAP1 LC3 family.

翻訳後修飾

The precursor molecule is cleaved by APG4B/ATG4B to form LC3-I. This is activated by
APG7L/ATG7, transferred to ATG3 and conjugated to phospholipid to form LC3-II.

細胞内局在

Cytoplasm > cytoskeleton. Endomembrane system. Cytoplasmic vesicle > autophagosome
membrane. LC3-II binds to the autophagic membranes.

画像2All lanes : Anti-LC3B antibody [EPR18709] - Autophagosome
Marker (ab192890) at 1/2000 dilution

Lane 1 : Wild-type HepG2 untreated control cell lysate
Lane 2 : Wild-type HepG2 Treated Chloroquine (50 uM, 16 h) celllysate

Lane 3 : MAP1LC3B knockout HepG2 untreated control cell lysate
Lane 4 : MAP1LC3B knockout HepG2 Treated Chloroquine (50uM, 16 h) cell lysate

Lane 5 : U-87 MG cell lysate
Lane 6 : PC-12 cell lysate
Western blot - Anti-LC3B antibody [EPR18709] Autophagosome Marker (ab192890)

Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.

Predicted band size: 15 kDa
Observed band size: 14,16 kDa

False colour image of Western blot: Anti-LC3B antibody[EPR18709] - Autophagosome Marker staining at 1/2000 dilution,
shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291)
loading control staining at 1/20000 dilution, shown in red. In
Western blot, ab192890 was shown to bind specifically to LC3B. Aband was observed at 16/14 kDa (yellow arrows) in treated wildtype HepG2 cell lysates with no signal observed at this size inMAP1LC3B knockout cell line ab277828 (knockout cell lysate

ab283796). To generate this image, wild-type and MAP1LC3B
knockout HepG2 cell lysates were analysed. First, samples wererun on an SDS-PAGE gel then transferred onto a nitrocellulose
membrane. Membranes were blocked in 3 % milk in TBS-0.1 %Tween® 20 (TBS-T) before incubation with primary antibodies
3overnight at 4 °C. Blots were washed four times in TBS-T,
incubated with secondary antibodies for 1 h at room temperature,
washed again four times then imaged. Secondary antibodies usedwere Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG
H&L 680RD at 1/20000 dilution.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: LC3B knockout HAP1 cell lysate (20 µg)
Lane 3: Human brain tissue lysate (20 µg)
Lane 4: U-87 MG cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green).
Green -target observed at 14 and 16 kDa. Red - loading control,ab8245, observed at 37 kDa.
This western blot image is a comparison between ab192890 and acompetitor's top cited rabbit polyclonal antibody.

Western blot - Anti-LC3B antibody [EPR18709] Autophagosome Marker (ab192890)

ab192890 staining LC3B in HeLa (Human epithelial cell line fromcervix adenocarcinoma) cells +/- Chloroquine (50μM, 24 hours).
The cells were fixed with 100% methanol (5 min), permeabilizedwith 0.1% Triton X-100 for 5 minutes and then blocked with 1%
BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for
1h. The cells were then incubated overnight at +4°C with ab192890
at 1μg/ml and ab195889, Mouse monoclonal to alpha Tubulin(Alexa Fluor® 594), at 1/250 dilution (shown in pseudocolor red)followed by a further incubation at room temperature for 1h with agoat secondary antibody to Rabbit IgG (Alexa Fluor® 488)
(ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labeled
Immunocytochemistry/ Immunofluorescence - AntiLC3B antibody [EPR18709] - Autophagosome
Marker (ab192890)

with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems,TCS SP8).
Alexa Fluor® 488 (ab225383) and Alexa Fluor® 647 (ab225382)
conjugated versions are available for this clone.

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ab192890 staining LC3B in paraffin embedded human astrocytoma
tissue by Immunohistochemistry. Heat mediated antigen retrieval
was performed with Citrate buffer (pH 6.0, Epitope Retrieval
Solution 1) for 20 mins. Samples were incubated with primary
antibody at 1/1000 dilution for 30 mins at room temperature. Readyto use LeicaDS9800 (Bond™ Polymer Refine Detection) was usedas the secondary antibody. Hematoxylin was used as acounterstain. The immunostaining was performed on a Leica
Biosystems BOND® RX instrument.

Immunohistochemistry (Formalin/PFA-fixed paraffinembedded sections) - Anti-LC3B antibody[EPR18709] - Autophagosome Marker (ab192890)

Immunohistochemical analysis of formalin fixed paraffin embedded
human cortex labelling LC3B with ab192890 at a dilution of 0.1µg/ml. The immunostaining was performed on a Ventana
DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument withan OptiView DAB IHC Detection Kit. Heat mediated antigen
retrieval was conducted for 32min with ULTRA cell conditioning
solution (CC1 pH8.5) . ab192890 anti LC3B antibody wasincubated at 37°C for 16min. Sections were counterstained is with
Hematoxylin II. Image inset shows absence of staining in secondary
antibody only controlImmunohistochemistry (Formalin/PFA-fixed paraffinembedded sections) - Anti-LC3B antibody[EPR18709] - Autophagosome Marker (ab192890)

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Tissue Microarrays stained for Anti-LC3B antibody [EPR18709] Autophagosome Marker using ab192890 in immunohistochemical
analysis. This table provides a detailed overview of positive (tickmark) and negaive (cross mark) staining per sample type tested.
Heat mediated antigen retrieval was performed with Citrate buffer(pH 6.0, Epitope Retrieval Solution 1) for 20 mins.
The section was incubated with ab192890 for 30 mins at room
temperature.
The immunostaining was performed on a Leica Biosystems
BOND® RX instrument.
Immunohistochemistry (Formalin/PFA-fixed paraffinembedded sections) - Anti-LC3B antibody[EPR18709] - Autophagosome Marker (ab192890)

IHC image of LC3B staining in a section of formalin-fixed paraffinembedded normal human cerebral cortex* performed on a Leica
BONDTM system using the standard protocol F. The section waspre-treated using heat mediated antigen retrieval with sodium
citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The
section was then incubated with ab221794, 0.1ug/ml, for 15 mins atroom temperature and detected using an HRP conjugated compact
polymer system. DAB was used as the chromogen. The section
was then counterstained with haematoxylin and mounted with DPX.
The inset secondary-only control image is taken from an identicalassay without primary antibody.
Immunohistochemistry (Formalin/PFA-fixed paraffin-

For other IHC staining systems (automated and non-automated)

embedded sections) - Anti-LC3B antibody

customers should optimize variable parameters such as antigen[EPR18709] - Autophagosome Marker (ab192890)

retrieval conditions, primary antibody concentration and antibodyincubation times.
This data was developed using the same antibody clone in adifferent buffer formulation containing only PBS (ab221794).

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ab192890 staining LC3B in HAP1 cells (wildtype and MAP1LC3Bknockout) +/- Chloroquine (50μM, 24 hours).
The cells were fixed with 100% methanol (5 min), permeabilizedwith 0.1% Triton X-100 for 5 minutes and then blocked with 1%
BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for
1h. The cells were then incubated overnight at +4°C with ab192890
at 1 μg/ml and ab195889, Mouse monoclonal to alpha Tubulin(Alexa Fluor® 594), at 1/250 dilution (shown in pseudocolor red)followed by a further incubation at room temperature for 1h with agoat secondary antibody to Rabbit IgG (Alexa Fluor® 488)
(ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labeled
Immunocytochemistry/ Immunofluorescence - AntiLC3B antibody [EPR18709] - Autophagosome
Marker (ab192890)

with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems,TCS SP8).

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: LC3B knockout HAP1 cell lysate (20 µg)
Lane 3: Human brain tissue lysate (20 µg)
Lane 4: U-87 MG cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab192890
observed at 14 and 16 kDa. Red - loading control, ab8245,observed at 37 kDa.

ab192890 was shown to specifically react with LC3B in wild-type
HAP1 cells. No band was observed when LC3B knockout samples
Western blot - Anti-LC3B antibody [EPR18709] Autophagosome Marker (ab192890)

were examined. Wild-type and LC3B knockout samples weresubjected to SDS-PAGE. ab192890 and ab8245 (loading controlto GAPDH) were diluted 1/2000 and 1/10,000 respectively andincubated overnight at 4°C. Blots were developed with Goat antiRabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and
Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed(ab216776) secondary antibodies at 1/10,000 dilution for 1 hour atroom temperature before imaging.

7All lanes : Anti-LC3B antibody [EPR18709] - Autophagosome
Marker (ab192890) at 1/2000 dilution

Lane 1 : Human brain lysate
Lane 2 : U-87 MG (Human glioblastoma-astrocytoma epithelial cellline) whole cell lysate

Lane 3 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 4 : RAW 264.7 (Mouse macrophage cell line transformedwith Abelson murine leukemia virus) whole cell lysate

Lane 5 : Mouse heart lysate
Lane 6 : Rat heart lysate
Western blot - Anti-LC3B antibody [EPR18709] -Lane 7 : Mouse brain lysate

Autophagosome Marker (ab192890)

Lane 8 : Rat brain lysate
Lysates/proteins at 20 µg per lane.

SecondaryAll lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to
the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 15 kDaObserved band size: 14,16 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure times: Lane 1-6: 3 minutes; Lane 7 and 8: 30 seconds.

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Different batches of ab192890 were tested onU-87 MG (Humanglioblastoma-astrocytoma epithelial cell line) lysate at 0.9 µg/ml. 15µg of lysate was loaded in each lane. Bands observed at 14,16kDa.

Western blot - Anti-LC3B antibody [EPR18709] Autophagosome Marker (ab192890)

Anti-LC3B antibody [EPR18709] - Autophagosome
Marker (ab192890)

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