Immunohistochemical analysis of paraffin-embedded human mammary gland tissue labeling CD133 with ab278053 at 1/1000 (0.5 µg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Apical staining on human mammary gland (PMID: 18261235). The section was incubated with ab278053 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling CD133 with ab278053 at 1/1000 (0.5 µg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on Bowman’s capsule of human kidney (PMID: 19875807). The section was incubated with ab278053 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Flow cytometric analysis of HEK-293T (Human embryonic kidney epithelial cell, Left panel) / Caco-2 (Human colorectal adenocarcinoma epithelial cell, Right panel ) using ab278053 at 1/500 dilution (0.1µg) (Red). Mouse monoclonal IgG  (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti mouse IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control: HEK-293T. (PMID 20167130)
Gated on viable cells.

All lanes : Anti-CD133 antibody [RM1002] (ab278053) at 1/1000 dilution

Lane 1 : Caco-2 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : HEK-293T (Human embryonic kidney epithelial cell) whole cell lysate
Lane 3 : NCCIT (Human pluripotent embryonic carcinoma epithelial cell) whole cell lysate
Lane 4 : HT-29 (Human colorectal adenocarcinoma epithelial cell) 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: 97 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?


Exposure time: 26 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Negative control: HEK-293T (PMID 20167130).

The MW observed is consistent with the literature (PMID 20717901; PMID 18645205).

CD133 was immunoprecipitated from 0.35 mg Caco-2 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate with ab278053 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab278053 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Caco-2 whole cell lysate 10 μg.

Lane 2: ab278053 IP in Caco-2 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab278053 in Caco-2 whole cell lysate.

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

Exposure time: 3 seconds.