Autophagy Analysis (ATG16L1, ATG16L1 pS278, SQSTM1, LC3B, Ubiquitin, M6PR) Antibody Sampler Panel (ab269811)

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: ATG16L1 knockout HAP1 cell lysate (20 µg) 
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Jurkat cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - Anti-ATG16L1 antibody [EPR15638] - N-terminal (ab187671) observed at 68 and 70 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab187671 was shown to recognize ATG16L1 when ATG16L1 knockout samples were used, along with additional cross-reactive bands. Wild-type and ATG16L1 knockout samples were subjected to SDS-PAGE. ab187671 and ab8245 (loading control to GAPDH) were diluted 1/2000 and 10000 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 1/10000 dilution for 1 h at room temperature before imaging.

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: SQSTM1 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: HepG2 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - Anti-SQSTM1 / p62 antibody [EPR4844] (ab109012) observed at 55 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab109012 was shown to specifically react with SQSTM1 in wild-type HAP1 cells. No band was observed when SQSTM1 knockout samples were used. Wild-type and SQSTM1 knockout samples were subjected to SDS-PAGE, Ab109012 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/10,000 dilution and 1/20,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20,000 dilution for 1 hour at room temperature before imaging.

Lane 1: Hap1 wildtype cell lysate (20 µg)
Lane 2: SQSTM1 Hap1 knockout cell lysate (20 µg)
Lane 3: HeLa wildtype cell lysate (20 µg)
Lane 4: SQSTM1 HeLa knockout cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - Anti-SQSTM1 / p62 antibody [EPR4844] (ab109012) observed at 64 kDa. Red - loading control, ab8245 observed at 37 kDa.
ab109012 was shown to react with SQSTM1 / p62 in HeLa wildtype. Loss of signal was observed when knockout sample ab263770 was used. Wild-type and SQSTM1 / p62 knockout samples were subjected to SDS-PAGE. ab109012 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4^°C at 1 in 10000 dilution and 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

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 Anti-LC3B antibody [EPR18709] (ab192890) and a competitor's top cited rabbit polyclonal antibody.

Primary: Anti-Ubiquitin antibody [EPR8830] (ab134953), Purified, 0.7 µg/mL

Lane 1: Rat brain lysate, 20 µg

Lane 2: Mouse kidney lysate, 20 µg

Lane 3: Rat kidney lysate, 20 µg

Secondary: ab97051, 1/20000 dilution

Blocking and diluting buffer: 5% NFDM/TBST.

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: M6PR (cation independent) knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: A549 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - Anti-M6PR (cation independent) antibody [EPR6599] (ab124767) observed at 274 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab124767 was shown to specifically react with M6PR (cation independent) in wild-type HAP1 cells as signal was lost in M6PR (cation independent) knockout cells. Wild-type and M6PR (cation independent) knockout samples were subjected to SDS-PAGE. Ab124767 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/50000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

Purified ab109012 staining SQSTM1 in wild-type HAP1 cells (top panel) and SQSTM1 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), 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 with ab109012 at 1μg/ml and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, 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 in blue with DAPI.

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

Immunohistochemical analysis of paraffin-embedded Human prostatic hyperplasia tissue labeling ATG16L1 with ab187671 at 1/100 dilution followed by pre-diluted HRP Polymer for Rabbit IgG secondary antibody and counter-stained with Hematoxylin.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

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).

Immunohistochemical staining of paraffin embedded mouse colon tissue section labelling M6PR with purified ab124767 at dilution of 1/500. The secondary antibody used was ab97051 Goat Anti-Rabbit IgG H&L (HRP), at a dilution of 1/500. The sample was counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset. 

Immunocytochemistry/ Immunofluorescence analysis of JAR (Human placenta choriocarcinoma cell line) cells labeling Ubiquitin with Purified ab134953 at 1:100 dilution (7.2μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

HCT116 wild-type and ATG16L1 knockout cells were incubated with either complete media or amino acid deficient DMEM for 3 hours. 5ug of whole cell lysate were resolved by SDS-PAGE on a 6%-18% gradient gel, then transferred onto PVDF membrane. Membrane was blocked in 10X blocking buffer (Cat # ab126587) diluted in TBS solution for 30 minutes; incubated with Anti-ATG16L1 (phospho S278) antibody [EPR19016] (ab195242)  at 1/1000 dilution in 2.5% BSA TBST solution overnight at 4°C ; incubated with 1/15000 secondary antibody in 2% milk TBST solution for 45 minutes. Immobilon ECL was applied for 1 minute then imaged with film.