DGKZ/DGK-zeta was immunoprecipitated from 0.35 mg of Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate with ab239080 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab239080 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.
Lane 1: Jurkat whole cell lysat 10 μg (input).
Lane 2: ab239080 IP in Jurkat whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab239080 in Jurkat whole cell lysate (-).

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 15 seconds.

 

Immunohistochemical analysis of paraffin-embedded rat brain tissue labeling DGKZ/DGK-zeta with ab239080 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic staining on neurons of rat brain (PMID: 14511325) (PMID: 24119575) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse brain tissue labeling DGKZ/DGK-zeta with ab239080 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic and nuclear staining on neurons of mouse brain (PMID: 14511325) (PMID: 24119575) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human cerebellum tissue labeling DGKZ/DGK-zeta with ab239080 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic and nuclear staining on Purkinje cells of human cerebellum (PMID: 14511325) (PMID: 24119575) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling DGKZ/DGK-zeta with ab239080 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Cytoplasmic staining on subset of immune cells in human tonsil (PMID: 24573202) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-DGKZ/DGK-zeta antibody [EPR22040-72] (ab239080) at 1/1000 dilution

Lane 1 : Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 2 : HuT-78 (human Sezary syndrome cutaneous T lymphocyte), whole cell lysate
Lane 3 : Human cerebellum tissue lysate
Lane 4 : Mouse brain tissue lysate
Lane 5 : Mouse cerebellum tissue lysate
Lane 6 : Mouse thymus tissue lysate
Lane 7 : Rat brain tissue lysate
Lane 8 : Rat cerebellum tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 124 kDa
Observed band size: 90-130 kDa why is the actual band size different from the predicted?

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

Exposure time:
Lane 1-2: 15 seconds.
Lane 3-8: 3 seconds.

The lysates were not boiled to prevent the target protein from aggregation. Multiple bands are likely due to isoforms and phosphorylation. The molecular mass of human DGKZ / DGK-zeta is also predicated to be larger than that of rodents. The molecular profile/weight observed is consistent with what has been described in the literature (PMID: 12070163, PMID: 14511325, PMID: 9657393).

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilzed Jurkat (human T cell leukemia cell line from peripheral blood) cell line labeling DGKZ/DGK-zeta with ab239080 at 1/600 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730, Black) isotype control, and an unlabeled control (Cell without incubation with primary antibody and secondary antibody, Blue).

Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 was used as the secondary antibody.