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.

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.

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.

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).

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 : 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 cell line) whole cell lysate
Lane 3 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 4 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 5 : Mouse heart lysate
Lane 6 : Rat heart lysate
Lane 7 : Mouse brain lysate
Lane 8 : Rat brain lysate

Lysates/proteins at 20 µg per lane.

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

Blocking/Dilution buffer: 5% NFDM/TBST.

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