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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Autophagy Analysis (ATG16L1, ATG16L1 pS278, SQSTM1, LC3B, Ubiquitin, M6PR) Antibody Sampler Panel (AB269811)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Autophagy Analysis (ATG16L1, ATG16L1 pS278, SQSTM1, LC3B, Ubiquitin, M6PR) Antibody Sampler Panel (AB269811)

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).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Autophagy Analysis (ATG16L1, ATG16L1 pS278, SQSTM1, LC3B, Ubiquitin, M6PR) Antibody Sampler Panel (AB269811)

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).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Autophagy Analysis (ATG16L1, ATG16L1 pS278, SQSTM1, LC3B, Ubiquitin, M6PR) Antibody Sampler Panel (AB269811)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Autophagy Analysis (ATG16L1, ATG16L1 pS278, SQSTM1, LC3B, Ubiquitin, M6PR) Antibody Sampler Panel (AB269811)

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.

• WB

Lab

Western blot - Autophagy Analysis (ATG16L1, ATG16L1 pS278, SQSTM1, LC3B, Ubiquitin, M6PR) Antibody Sampler Panel (AB269811)

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.

Lane 1:

Western blot - Anti-M6PR (cation independent) antibody [EPR6599] - Lysosome Membrane Marker (<a href='/en-us/products/primary-antibodies/m6pr-cation-independent-antibody-epr6599-lysosome-membrane-marker-ab124767'>ab124767</a>)

Lanes 2 - 4:

Western blot - Anti-M6PR (cation independent) antibody [EPR6599] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/m6pr-cation-independent-antibody-epr6599-bsa-and-azide-free-ab226090'>ab226090</a>) at 1/50000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

M6PR (cation independent) knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

A549 whole cell lysate at 20 µg

Predicted band size: 274 kDa

false

• WB

Collaborator

Western blot - Autophagy Analysis (ATG16L1, ATG16L1 pS278, SQSTM1, LC3B, Ubiquitin, M6PR) Antibody Sampler Panel (AB269811)

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.

All lanes:

Western blot - Anti-ATG16L1 (phospho S278) antibody [EPR19016] (<a href='/en-us/products/primary-antibodies/atg16l1-phospho-s278-antibody-epr19016-ab195242'>ab195242</a>)

Predicted band size: 68 kDa

true

This image is courtesy of Dr Ryan Russell (University of Ottawa).

• WB

Lab

Western blot - Autophagy Analysis (ATG16L1, ATG16L1 pS278, SQSTM1, LC3B, Ubiquitin, M6PR) Antibody Sampler Panel (AB269811)

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.

All lanes:

Western blot - Anti-LC3B antibody [EPR18709] - Autophagosome Marker (<a href='/en-us/products/primary-antibodies/lc3b-antibody-epr18709-autophagosome-marker-ab192890'>ab192890</a>)

Predicted band size: 15 kDa

false

• WB

Lab

Western blot - Autophagy Analysis (ATG16L1, ATG16L1 pS278, SQSTM1, LC3B, Ubiquitin, M6PR) Antibody Sampler Panel (AB269811)

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.

All lanes:

Western blot - Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (<a href='/en-us/products/primary-antibodies/sqstm1-p62-antibody-epr4844-autophagosome-marker-ab109012'>ab109012</a>) at 1/10000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

SQSTM1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

HepG2 whole cell lysate at 20 µg

Predicted band size: 47 kDa

false

• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-ATG16L1 antibody [EPR15638] - N-terminal (<a href='/en-us/products/primary-antibodies/atg16l1-antibody-epr15638-n-terminal-ab187671'>ab187671</a>) at 1/2000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

ATG16L1 knockout HAP1 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Predicted band size: 68 kDa

false

• WB

Unknown

Western blot - Autophagy Analysis (ATG16L1, ATG16L1 pS278, SQSTM1, LC3B, Ubiquitin, M6PR) Antibody Sampler Panel (AB269811)

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.

All lanes:

Western blot - Anti-Ubiquitin antibody [EPR8830] (<a href='/en-us/products/primary-antibodies/ubiquitin-antibody-epr8830-ab134953'>ab134953</a>) at 0.7 µg/mL

Lane 1:

Rat brain lysate at 20 µg

Lane 2:

Mouse kidney lysate at 20 µg

Lane 3:

Rat kidney lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 26 kDa

Observed band size: 8 kDa

false

• WB

Supplier Data

Western blot - Autophagy Analysis (ATG16L1, ATG16L1 pS278, SQSTM1, LC3B, Ubiquitin, M6PR) Antibody Sampler Panel (AB269811)

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.

false