Anti-LC3B antibody (ab63817)

All lanes : Anti-LC3B antibody (ab63817) at 1 µg/ml

Lane 1 : Wild-type HepG2 untreated control cell lysate
Lane 2 : Wild-type HepG2 Treated Chloroquine (50 uM, 16 h) cell lysate
Lane 3 : MAP1LC3B knockout HepG2 untreated control cell lysate
Lane 4 : MAP1LC3B knockout HepG2 Treated Chloroquine (50 uM, 16 h) cell lysate
Lane 5 : U-87 MG cell lysate
Lane 6 : PC-12 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 15 kDa
Observed band size: 14,16 kDa why is the actual band size different from the predicted?

False colour image of Western blot: Anti-LC3B antibody staining at 1 ug/ml, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab63817 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.

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: U87MG cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab63817 observed at 16 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab63817 was shown to recognize LC3B in wild-type HAP1 cells along with additional cross reactive bands. No band was observed when LC3B knockout samples were examined. Wild-type and LC3B knockout samples were subjected to SDS-PAGE. ab63817 and ab8245 (loading control to GAPDH) were diluted 1 µg/mL 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.

ab63817 staining LC3B in HeLa 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 ab63817 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 labelled with DAPI (shown in blue).

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

ab63817 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 ab63817 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 labelled with DAPI (shown in blue).

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

IHC image of LC3B staining in human hippocampus formalin fixed paraffin embedded tissue section, 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 20 mins. The section was then incubated with ab63817, 1µg/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.

Anti-LC3B antibody (ab63817) at 1 µg/ml + Human brain tissue lysate - total protein (ab29466) at 10 µg

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 15 kDa
Observed band size: 15 kDa
Additional bands at: 32 kDa, 54 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 2 minutes

All lanes : Anti-LC3B antibody (ab63817) at 1 µg/ml

Lane 1 : Brain (Mouse) Tissue Lysate
Lane 2 : Brain (Rat) Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 15 kDa
Observed band size: 15 kDa
Additional bands at: 30 kDa, 65 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 3 minutes

Anti-LC3B antibody (ab63817) at 1 µg/ml + LC3B protein (Human) (ab87944) at 0.1 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 15 kDa


Exposure time: 2 minutes

Immunocytochemical analysis of HeLa cells, labeling LC3B with ab63817 (1/200 with PBS for 16 hours at 22°C). Cells were methanol fixed, immunofluorescently labeled, adn counterstained with DAPI. 

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