All lanes : Anti-Cyclin T1 antibody [EPR17982] (ab184703) at 1/1000 dilution

Lane 1 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 4 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 5 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 81 kDa
Observed band size: 81 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Ab184703 staining Cyclin T1 in Jurkat (human T cell leukemia T lymphocyte). Cells were fixed with 100% Methanol. Samples were incubated with primary antibody at 1/100 dilution (6.4µg/ml). An Alexa Fluor^® 488 Goat anti-rabbit (ab150077) was used as the secondary antibody at 1/1000 dilution (2 μg/ml). Ab195888, anti-alpha Tubulin [DM1A] – Microtubule Marker (Alexa Fluor^® 594) was used as counterstain antibody at 1/200 dilution (2.5 μg/ml). DAPI was used as a nuclear counterstain. Confocal image showing nuclear staining in Jurkat cell line.

Ab184703 staining Cyclin T1 in MCF7 (human breast adenocarcinoma epithelial cell). Cells were fixed with 100% Methanol. Samples were incubated with primary antibody at 1:100 dilution (6.4 μg/ml). An Alexa Fluor^® 488 Goat anti-rabbit (ab150077) was used as the secondary antibody at 1/1000 dilution (2 μg/ml). Ab195888, anti-alpha Tubulin [DM1A] – Microtubule Marker (Alexa Fluor^® 594) was used as counterstain antibody at 1/200 dilution (2.5 μg/ml). DAPI was used as a nuclear counterstain. Confocal image showing nuclear staining in MCF7 cell line.

All lanes : Anti-Cyclin T1 antibody [EPR17982] (ab184703) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse kidney lysate
Lane 3 : Mouse spleen lysate
Lane 4 : Rat brain lysate
Lane 5 : Rat spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 81 kDa
Observed band size: 81 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1,2 and 3: 15 seconds; Lane 4: 3 minutes.

The antibody did not detect the target protein from human tissues (WB or IHC) but the IHC application is recommended for mouse and rat. In addition the failure of human tissue WB might result from shortage of proper human tissue.

All lanes : Anti-Cyclin T1 antibody [EPR17982] (ab184703) at 1/1000 dilution

Lane 1 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 2 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 4 : NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 81 kDa
Observed band size: 81 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 3 minutes; Lane 2, 3 and 4: 15 seconds.

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Cyclin T1 with ab184703 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nucleus staining on hepatocytes of mouse liver 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) (ab97051) at 1/500 dilution.

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

Immunohistochemical analysis of paraffin-embedded Rat kidney tissue labeling Cyclin T1 with ab184703 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nucleus staining on rat kidney 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) (ab97051) at 1/500 dilution.

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

Flow cytometric analysis of 4% paraformaldehyde-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling Cyclin T1 with ab184703 at 1/120 dilution (red) compared with a Rabbit IgG,monoclonal [EPR17982] -  Isotype Control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/500 dilution was used as the secondary antibody.

Cyclin T1 was immunoprecipitated from 1mg of PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate with ab184703 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab184703 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.
Lane 1: PC-12 whole cell lysate 10µg (Input).
Lane 2: ab184703 IP in PC-12 whole cell lysate.
Lane 3: Rabbit IgG,monoclonal [EPR17982] - Isotpe Control (ab172730) instead of ab184703 in PC-12 whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 30 seconds.