Autophagy Analysis (ATG16L1, ATG16L1 pS278, SQSTM1, LC3B, Ubiquitin, M6PR) Antibody Sampler Panel (ab269811) is part of the multiplex kits range. Abcam offers high-quality biological reagents and tools including antibodies, proteins, assays, cell lines and lysates.
Human, Mouse, Rat
ATG16L1
Application | Reactivity | Dilution info | Notes |
---|---|---|---|
Application ICC/IF | Reactivity Reacts | Dilution info - | Notes Anti-ATG16L1 antibody [EPR15638] - N-terminal ab187671 has not been in house tested for ICC/IF. |
Application IHC-P | Reactivity Reacts | Dilution info - | Notes Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker ab109012 has not been in house tested for IHC-P. |
Application WB | Reactivity Reacts | Dilution info - | Notes For optimal WB results using Anti-ATG16L1 (phospho S278) antibody [EPR19016] ab195242, we recommend blocking with 10X Blocking Buffer (10X Blocking Buffer ab126587) |
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Plays an essential role in both canonical and non-canonical autophagy: interacts with ATG12-ATG5 to mediate the lipidation to ATG8 family proteins (MAP1LC3A, MAP1LC3B, MAP1LC3C, GABARAPL1, GABARAPL2 and GABARAP) (PubMed:23376921, PubMed:23392225, PubMed:24553140, PubMed:24954904, PubMed:27273576, PubMed:29317426, PubMed:30778222, PubMed:33909989). Acts as a molecular hub, coordinating autophagy pathways via distinct domains that support either canonical or non-canonical signaling (PubMed:29317426, PubMed:30778222). During canonical autophagy, interacts with ATG12-ATG5 to mediate the conjugation of phosphatidylethanolamine (PE) to ATG8 proteins, to produce a membrane-bound activated form of ATG8 (PubMed:23376921, PubMed:23392225, PubMed:24553140, PubMed:24954904, PubMed:27273576). Thereby, controls the elongation of the nascent autophagosomal membrane (PubMed:23376921, PubMed:23392225, PubMed:24553140, PubMed:24954904, PubMed:27273576). As part of the ATG8 conjugation system with ATG5 and ATG12, required for recruitment of LRRK2 to stressed lysosomes and induction of LRRK2 kinase activity in response to lysosomal stress (By similarity). Also involved in non-canonical autophagy, a parallel pathway involving conjugation of ATG8 proteins to single membranes at endolysosomal compartments, probably by catalyzing conjugation of phosphatidylserine (PS) to ATG8 (PubMed:33909989). Non-canonical autophagy plays a key role in epithelial cells to limit lethal infection by influenza A (IAV) virus (By similarity). Regulates mitochondrial antiviral signaling (MAVS)-dependent type I interferon (IFN-I) production (PubMed:22749352, PubMed:25645662). Negatively regulates NOD1- and NOD2-driven inflammatory cytokine response (PubMed:24238340). Instead, promotes an autophagy-dependent antibacterial pathway together with NOD1 or NOD2 (PubMed:20637199). Plays a role in regulating morphology and function of Paneth cell (PubMed:18849966).
APG16L, UNQ9393/PRO34307, ATG16L1, Autophagy-related protein 16-1, APG16-like 1
Autophagy Analysis (ATG16L1, ATG16L1 pS278, SQSTM1, LC3B, Ubiquitin, M6PR) Antibody Sampler Panel (ab269811) is part of the multiplex kits range. Abcam offers high-quality biological reagents and tools including antibodies, proteins, assays, cell lines and lysates.
Human, Mouse, Rat
ATG16L1
ICC/IF, IHC-P, WB
Blue Ice
-20°C
-20°C
-20°C
Please note that Recombinant Anti-ATG16L1 (phospho S278) antibody [EPR19016] is for mouse and human reactivity only. The rest of the clones in this panel react with human, mouse, and rat.
Autophagy Analysis (ATG16L1, ATG16L1 pS278, SQSTM1, LC3B, Ubiquitin, M6PR) Antibody Sampler Panel ab269811 contains multiple trial-sized versions of anti-human, mouse and rat antibody clones against ATG16L1, ATG16L1 pS278, SQSTM1, LC3B, Ubiquitin and M6PR, specifically selected for high performance in in various applications. This panel contains 6 recombinant rabbit monoclonal antibodies against human, mouse and rat ATG16L1, ATG16L1 pS278, SQSTM1, LC3B, Ubiquitin and M6PR. They are provided as a sampler panel to allow you to easily evaluate each antibody.
For guidelines on how to use each antibody within the panel, please consult the individual datasheet for each antibody.
Panel contains:
Recombinant Anti-ATG16L1 (phospho S278) antibody [EPR19016] (20 μL) ab195242
Recombinant Anti-ATG16L1 antibody [EPR15638] - N-terminal antibody (20 μL) ab187671
Recombinant Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker antibody (20 μL) ab109012
Recombinant Anti-LC3B antibody [EPR18709] - Autophagosome Marker antibody (20 μL) ab192890
Recombinant Anti-Ubiquitin antibody [EPR8830] (20 μL) ab134953
Recombinant Anti-M6PR (cation independent) antibody [EPR6599] - Lysosome Membrane Marker (20 μL) ab124767
Please note that Recombinant Anti-ATG16L1 (phospho S278) antibody [EPR19016] is for mouse reactivity only. The rest of the clones in this panel react with human, mouse, and rat.
For optimal WB results using ab195242, we recommend blocking with 10X Blocking Buffer (ab126587).
Explore our range of antibody sample panels designed to provide you with a variety of trial-size antibodies in a convenient and cost-effective format.
Carrier-free formulations of our recombinant antibodies are also available for easy conjugation to labels of your choice and for multiplex applications. Use our intuitive search and select carrier-free or your label of choice. For bespoke conjugations or large volumes email bespoke@abcam.com.
This supplementary information is collated from multiple sources and compiled automatically.
Ubiquitin acts as a small regulatory protein with a mass of around 8.5 kDa. It is widely expressed in eukaryotic cells and involved in tagging cellular proteins for degradation. M6PR or cation-independent mannose-6-phosphate receptor functions in the lysosomal targeting of proteins aiding in lysosomal enzyme transport. SQSTM1 commonly known as p62 plays an important role in autophagy by linking ubiquitinated proteins to the autophagy machinery. ATG16L1 essential for autophagy participates in the formation of autophagosomes. LC3B a marker of autophagy associates with autophagosomes and its lipidation signifies autophagic activity.
These proteins serve central roles in cellular homeostasis and degradation processes. Ubiquitin is integral in tagging proteins for proteasomal degradation. M6PR functions within lysosomal enzyme trafficking while p62 aggregates ubiquitinated proteins for autophagic degradation acting as a bridge to autophagic cargo. ATG16L1 stands as part of the autophagy-related gene complex important for the elongation of the autophagosome membrane. LC3B when lipidated embeds into autophagosome membranes distinguishing active autophagy processes.
These proteins integrate into the ubiquitin-proteasome and autophagy-lysosome systems. Ubiquitin interacts with various cellular pathways to manage protein turnover and degradation. The recycling of cellular components through autophagy involves p62 and ATG16L1 where LC3B serves as an autophagosome marker. These pathways intersect with cellular stress responses involving other proteins like mTOR and ULK1 which regulate autophagy initiation.
These proteins link to neurodegenerative diseases and cancer. Ubiquitin and related pathways play a role in conditions like Parkinson’s disease where impaired protein degradation is a factor. Altered expression of p62 or ATG16L1 can contribute to cancer progression through dysregulated autophagy. Both proteins interact with pathways affecting diseases with aberrant ubiquitin signaling or p62 accumulation associated with pathological states.
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Terms & Conditions.
Immunocytochemistry/ Immunofluorescence analysis of JAR (Human placenta choriocarcinoma cell line) cells labeling Ubiquitin with Purified Anti-Ubiquitin antibody [EPR8830] 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 Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 μg/ml). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) 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.
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] (Anti-M6PR (cation independent) antibody [EPR6599] ab124767) observed at 274 kDa. Red - loading control, ab18058, observed at 130 kDa.
Anti-M6PR (cation independent) antibody [EPR6599] 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. Anti-M6PR (cation independent) antibody [EPR6599] 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 Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed 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] (Anti-M6PR (cation independent) antibody [EPR6599] ab124767)
Lanes 2 - 4: Western blot - Anti-M6PR (cation independent) antibody [EPR6599] - BSA and Azide free (Anti-M6PR (cation independent) antibody [EPR6599] - BSA and Azide free ab226090) 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
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, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
This western blot image is a comparison between Anti-LC3B antibody [EPR18709] (Anti-LC3B antibody [EPR18709] - Autophagosome Marker ab192890) and a competitor's top cited rabbit polyclonal antibody.
All lanes: Western blot - Anti-LC3B antibody [EPR18709] - Autophagosome Marker (Anti-LC3B antibody [EPR18709] - Autophagosome Marker ab192890)
Predicted band size: 15 kDa
Primary: Anti-Ubiquitin antibody [EPR8830] (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: Goat Anti-Rabbit IgG H&L (HRP) ab97051, 1/20000 dilution
Blocking and diluting buffer: 5% NFDM/TBST.
All lanes: Western blot - Anti-Ubiquitin antibody [EPR8830] (Anti-Ubiquitin antibody [EPR8830] ab134953) 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
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Predicted band size: 26 kDa
Observed band size: 8 kDa
Blocking and diluting buffer: 5% NFDM/TBST
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] (Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker ab109012) observed at 64 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245 observed at 37 kDa.
Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker ab109012 was shown to react with SQSTM1 / p62 in HeLa wildtype. Loss of signal was observed when knockout sample Human SQSTM1 (p62) knockout HEK-293T cell lysate ab263770 was used. Wild-type and SQSTM1 / p62 knockout samples were subjected to SDS-PAGE. Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker ab109012 and Anti-GAPDH antibody [6C5] - Loading Control (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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (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.
Immunohistochemical analysis of paraffin-embedded Human prostatic hyperplasia tissue labeling ATG16L1 with Anti-ATG16L1 antibody [EPR15638] - N-terminal 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.
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] (Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker ab109012) observed at 55 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker 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, Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker ab109012 and Anti-GAPDH antibody [6C5] - Loading Control 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 Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed 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 (Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker ab109012) 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
Purified Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker 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 Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker ab109012 at 1μg/ml and Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed 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 staining of paraffin embedded mouse colon tissue section labelling M6PR with purified Anti-M6PR (cation independent) antibody [EPR6599] ab124767 at dilution of 1/500. The secondary antibody used was Goat Anti-Rabbit IgG H&L (HRP) 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.
LC3B immunofluorescence staining of HAP1 cells using rabbit anti-LC3B antibody
Anti-LC3B antibody [EPR18709] - Autophagosome Marker 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 Anti-LC3B antibody [EPR18709] - Autophagosome Marker ab192890 at 1 μg/ml and Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed 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).
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 # 10X Blocking Buffer ab126587) diluted in TBS solution for 30 minutes; incubated with Anti-ATG16L1 (phospho S278) antibody [EPR19016] (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 - Recombinant Anti-ATG16L1 (phospho S278) antibody [EPR19016] (Anti-ATG16L1 (phospho S278) antibody [EPR19016] ab195242)
Developed using the ECL technique.
Predicted band size: 68 kDa
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 (Anti-ATG16L1 antibody [EPR15638] - N-terminal ab187671) observed at 68 and 70 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
Anti-ATG16L1 antibody [EPR15638] - N-terminal 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. Anti-ATG16L1 antibody [EPR15638] - N-terminal ab187671 and Anti-GAPDH antibody [6C5] - Loading Control 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (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 (Anti-ATG16L1 antibody [EPR15638] - N-terminal ab187671) 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
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