Anti-ATG14 antibody [EPR26188-66]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-ATG14 antibody [EPR26188-66] (AB315009)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized ATG14 KO HeLa (human ATG14 knockout cervical adenocarcinoma epithelial cell, Left) / Parental HeLa (Right) cells labelling ATG14 with ab315009 at 1/50 dilution (1 ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• WB

Supplier Data

Western blot - Anti-ATG14 antibody [EPR26188-66] (AB315009)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

ab315009 can detect both ATG14L and ATG14S isoforms (PMID : 36371383).

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

All lanes:

Western blot - Anti-ATG14 antibody [EPR26188-66] (ab315009) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell 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

Observed band size: 58 kDa,65 kDa

false

Exposure time: 59s

• WB

Supplier Data

Western blot - Anti-ATG14 antibody [EPR26188-66] (AB315009)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

ab315009 can detect both ATG14L and ATG14S isoforms (PMID : 36371383).

In Western blot, ab315009 was shown to bind specifically to ATG14. Target of interest was observed at 65 kDa and 58 KDa in wild-type HeLa cell lysates (lane 1) with no signal observed at this size in ATG14 knockout cell line (lane 2) (lane 2, knockout cell line ab264684 / knockout cell lysate ab258319).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-ATG14 antibody [EPR26188-66] (ab315009) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

ATG14 knockout HeLa whole cell 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

Observed band size: 65 kDa,58 kDa,36 kDa

false

Exposure time: 81s

• WB

Supplier Data

Western blot - Anti-ATG14 antibody [EPR26188-66] (AB315009)

Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of TBS.

ab315009 can detect both ATG14L and ATG14S isoforms (PMID : 36371383).

In lanes 2 and 4, the lysate was stored at -80℃ prior to Western Blotting. In lanes 1 and 3, lysate was freshly made and used for Western Blotting immediately to minimize protein degradation.

The samples were run on a Bis-Tris gel under reducing conditions.

Anti-ATG14 antibody (ab315009) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody 6C5 (ab8245) loading control staining at 1/20000 dilution, shown in red.

All lanes:

Western blot - Anti-ATG14 antibody [EPR26188-66] (ab315009) at 1/1000 dilution

Lane 1:

Fresh HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Frozen HCT 116 whole cell lysate at 20 µg

Lane 3:

Fresh HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Frozen HepG2 whole cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat Anti-Rabbit IgG H&L (800CW) at 1/20000 dilution

Lanes 1 - 4:

Goat Anti-Mouse IgG H&L (680RD) at 1/20000 dilution

Observed band size: 58 kDa,65 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-ATG14 antibody [EPR26188-66] (AB315009)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

ab315009 can detect both ATG14L and ATG14S isoforms (PMID : 36371383).

All lanes:

Western blot - Anti-ATG14 antibody [EPR26188-66] (ab315009) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse testis tissue lysate at 20 µg

Lane 3:

Mouse pancreas tissue lysate at 20 µg

Lane 4:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 5:

Rat brain tissue lysate at 20 µg

Lane 6:

Rat testis tissue 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

Observed band size: 58 kDa,65 kDa

false

Exposure time: 81s