Anti-LC3B 抗体 [EPR18709] - Autophagosome Marker

• WB

Lab

Western blot - Anti-LC3B antibody [EPR18709] - Autophagosome Marker (AB192890)

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 a competitor's top cited rabbit polyclonal antibody.

All lanes:

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

Predicted band size: 15 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LC3B antibody [EPR18709] - Autophagosome Marker (AB192890)

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. Ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used as the secondary antibody. Hematoxylin was used as a counterstain. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LC3B antibody [EPR18709] - Autophagosome Marker (AB192890)

IHC image of LC3B staining in a section of formalin-fixed paraffin-embedded normal human cerebral cortex* performed on a Leica BOND^TM system using the standard protocol F. The section was pre-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 at room 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 identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

This data was developed using the same antibody clone in a different buffer formulation containing only PBS (ab221794).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-LC3B antibody [EPR18709] - Autophagosome Marker (AB192890)

ab192890 staining LC3B in HAP1 cells (wildtype and MAP1LC3B knockout) +/- Chloroquine (50μM 24 hours).

The cells were fixed with 100% methanol (5 min) permeabilized with 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 a goat secondary antibody to Rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labeled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-LC3B antibody [EPR18709] - Autophagosome Marker (AB192890)

ab192890 staining LC3B in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells +/- Chloroquine (50μM 24 hours).

The cells were fixed with 100% methanol (5 min) permeabilized with 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 a goat secondary antibody to Rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labeled 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded 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 0.1 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an 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 was incubated at 37°C for 16min. Sections were counterstained is with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control

• IP

Supplier Data

Immunoprecipitation - Anti-LC3B antibody [EPR18709] - Autophagosome Marker (AB192890)

LC3B was immunoprecipitated from 1 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab192890 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab192890 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.
Lane 1 : HeLa whole cell lysate 10 μg (Input).
Lane 2 : ab192890 IP in HeLa whole cell lysate.
Lane 3 : Rabbit IgG,monoclonal [EPR25A] Isotype Control (ab172730) instead of ab192890 in HeLa whole cell cell lysate.
Blocking/Dilution buffer : 5% NFDM/TBST.
Exposure time : 30 seconds.

All lanes:

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

Predicted band size: 15 kDa

false

Exposure time: 30s

• WB

Lab

Western blot - Anti-LC3B antibody [EPR18709] - Autophagosome Marker (AB192890)

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. A band was observed at 16/14 kDa (yellow arrows) in treated wild-type HepG2 cell lysates with no signal observed at this size in MAP1LC3B knockout cell line ab277828 (knockout cell lysate ab283796). To generate this image, wild-type and MAP1LC3B knockout HepG2 cell lysates were analysed. First, samples were run 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 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 used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-LC3B antibody [EPR18709] - Autophagosome Marker (ab192890) at 1/1000 dilution

Lane 1:

Wild-type HepG2 untreated control cell lysate at 20 µg

Lane 2:

Wild-type HepG2 Treated Chloroquine (50 uM, 16 h) cell lysate at 20 µg

Lane 2:

Western blot - Human MAP1LC3B knockout Hep G2 cell line (ab277828)

Lane 2:

Western blot - Human MAP1LC3B knockout Hep G2 cell lysate (ab283796)

Lane 3:

MAP1LC3B knockout HepG2 untreated control cell lysate at 20 µg

Lane 4:

MAP1LC3B knockout HepG2 Treated Chloroquine (50 uM, 16 h) cell lysate at 20 µg

Lane 5:

U-87 MG cell lysate at 20 µg

Lane 6:

PC-12 cell lysate at 20 µg

Predicted band size: 15 kDa

Observed band size: 14 kDa,16 kDa

false

• WB

Lab

Western blot - Anti-LC3B antibody [EPR18709] - Autophagosome Marker (AB192890)

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

All lanes:

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

Predicted band size: 15 kDa

false

• WB

Lab

Western blot - Anti-LC3B antibody [EPR18709] - Autophagosome Marker (AB192890)

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 were examined. Wild-type and LC3B knockout samples were subjected to SDS-PAGE. ab192890 and ab8245 (loading control to GAPDH) were diluted 1/2000 and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit 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 at room temperature before imaging.

All lanes:

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

Predicted band size: 15 kDa

false

• WB

Supplier Data

Western blot - Anti-LC3B antibody [EPR18709] - Autophagosome Marker (AB192890)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure times : Lane 1-6 : 3 minutes; Lane 7 and 8 : 30 seconds.

All lanes:

Western blot - Anti-LC3B antibody [EPR18709] - Autophagosome Marker (ab192890) at 1/2000 dilution

Lane 1:

Human brain lysate at 20 µg

Lane 2:

U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate at 20 µg

Lane 3:

C6 (Rat glial tumor cell line) whole cell lysate at 20 µg

Lane 4:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 20 µg

Lane 5:

Mouse heart lysate at 20 µg

Lane 6:

Rat heart lysate at 20 µg

Lane 7:

Mouse brain lysate at 20 µg

Lane 8:

Rat brain lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 15 kDa

Observed band size: 14 kDa,16 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LC3B antibody [EPR18709] - Autophagosome Marker (AB192890)

Tissue Microarrays stained for Anti-LC3B antibody [EPR18709] - Autophagosome Marker using ab192890 in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) 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.