Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR19016] to ATG16L1 (phospho S278)
- Suitable for: ICC/IF, IHC-P, Dot blot, WB
- Reacts with: Mouse, Human
Product nameAnti-ATG16L1 (phospho S278) antibody [EPR19016]
See all ATG16L1 primary antibodies
DescriptionRabbit monoclonal [EPR19016] to ATG16L1 (phospho S278)
Tested applicationsSuitable for: ICC/IF, IHC-P, Dot blot, WBmore details
Species reactivityReacts with: Mouse, Human
Synthetic peptide within Mouse ATG16L1 aa 250-350 (phospho S278). The exact sequence is proprietary. Produced with two peptides within this region.
Database link: Q8C0J2
- WB: HEK-293 overexpressing ATG16L1 (WT) whole cell lysate. IHC-P: Human muscle tissue.
Co-immunization performed with both peptides, clone obtained by screening with peptide 1.
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
Storage bufferpH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 0.05% BSA, 40% Glycerol, PBS
Concentration information loading...
PurityProtein A purified
Our Abpromise guarantee covers the use of ab195242 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||Use a concentration of 3 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.|
|WB||Use at an assay dependent concentration. Predicted molecular weight: 68 kDa.
For optimal WB signal, we recommend using 10X Blocking Buffer (ab126587).
FunctionPlays an essential role in autophagy: interacts with ATG12-ATG5 to mediate the conjugation of phosphatidylethanolamine (PE) to LC3 (MAP1LC3A, MAP1LC3B or MAP1LC3C), to produce a membrane-bound activated form of LC3 named LC3-II. Thereby, controls the elongation of the nascent autophagosomal membrane.
Involvement in diseaseInflammatory bowel disease 10
Sequence similaritiesBelongs to the WD repeat ATG16 family.
Contains 7 WD repeats.
modificationsProteolytic cleavage by activated CASP3 leads to degradation and may regulate autophagy upon cellular stress and apoptotic stimuli.
Cellular localizationCytoplasm. Preautophagosomal structure membrane. Recruited to omegasomes membranes by WIPI2. Omegasomes are endoplasmic reticulum connected strutures at the origin of preautophagosomal structures. Localized to preautophagosomal structure (PAS) where it is involved in the membrane targeting of ATG5. Localizes also to discrete punctae along the ciliary axoneme.
- Information by UniProt
FormThere are 4 isoforms produced by alternative splicing.
- A16L1_HUMAN antibody
- APG16 like 1 antibody
- APG16-like 1 antibody
IHC images of vessel staining of ab195242, ATG16L1 (phospho S278), in sections of formalin-fixed paraffin-embedded normal human skeletal muscle tissue*, performed on a Leica BONDTM system using a modified protocol F.
Both sections were pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. One section was then treated with 200 enzyme units of alkaline phosphatase (AP+) for 1 hour at 37°C; and the other in buffer containing no alkaline phosphatase (AP-) for 1 hour at 37°C. The sections were then incubated with ab195242, 3µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The sections were then counterstained with haematoxylin and mounted with DPX.
Identical assays were also performed using detection system-only (no primary antibody) as reagent controls (data not shown), to ensure that staining seen was a result of the binding of the primary antibody.
The absence of staining in the AP+ tissue compared to the AP- tissue adds further evidence of phospho specificity for this antibody.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
IHC images of mice quadricep showing either pATG16L1 or LC3B staining
Mice were fed ad libitum or starved for 16 hours. Quadricep muscle were immediately harvested and fixed in 10% formalin for 2 days. The samples were then paraffin embedded, sectioned into 4µm thick slices, and mounted onto glass microscope slides. Slides were stained with primary antibody overnight at 4˚C: LC3B 1/1000, pATG16L1 (ab195242) 1/300. Secondary antibody: Alexa Fluor 555 anti-rabbit, 1/1000.
IF showing pATG16L1 (red) and total ATG16L1 (green):
Polyclonal population of ATG16L1 KO and HA-ATG16L1 reconstituted cells were starved of amino acid for 1 hour and stained. Blocking buffer used for pATG16L1 staining: 0.1% BSA, 1x abcam blocking buffer ab126587, diluted in PBS. Anti-pATG16L1 (ab195242) concentration: 1/150. Anti-ATG16L1, concentration: 1/200 Secondary antibody (Alexa Fluor 647/488) concentration: 1/1000.
IF showing pATG16L1 (red), LC3B (green) and p62 (white):
MEF cells were amino acid starved for 1 hour. Blocking buffer used for pATG16L1 (ab195242): 0.1% BSA, 1x abcam blocking buffer (ab126587), diluted in PBS. Anti-pATG16L1 (ab195242) concentration: 1/150. Secondary antibody (Alexa Fluor 647) concentration: 1/1000
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 1:1000 primary antibody 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 : Anti-ATG16L1 (phospho S278) antibody [EPR19016] (ab195242) at 1/1000 dilution
Lane 1 : HEK-293 (human epithelial cell line from embryonic kidney) transfected with an empty vector (vector control), containing a myc-His-tag®, whole cell lysate
Lane 2 : HEK-293 (human epithelial cell line from embryonic kidney) transfected with ATG16L1 (WT) expression vector containing a myc-His-tag®, whole cell lysate
Lane 3 : HEK-293 (human epithelial cell line from embryonic kidney) transfected with ATG16L1 (WT) expression vector containing a myc-His-tag®, followed by treatment with alkaline phosphatase for 1 hour, whole cell lysate
Lane 4 : HEK-293 (human epithelial cell line from embryonic kidney) transfected with ATG16L1 S278A expression vector containing a myc-His-tag®, whole cell lysate
Lysates/proteins at 10 µg per lane.
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Developed using the ECL technique.
Predicted band size: 68 kDa
Observed band size: 68 kDa
Exposure time: 3 minutes
Blocking/Dilution buffer: 5% NFDM/TBST.
Dot blot analysis of ATG16L1 (phospho S278) labeled with ab195242 at 1/1,000 dilution.
Lane 1: Mouse ATG16L1 (phospho S278) peptide;
Lane 2: Mouse ATG16L1 non-phospho peptide;
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100,000 dilution was used as secondary antibody.
Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 3 minutes.
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
ab195242 has been referenced in 2 publications.