All lanes : Anti-CD133 antibody [EPR20980-104] (ab216323) at 1/1000 dilution

Lane 1 : Caco-2 cell lysate
Lane 2 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 97 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?


Exposure time: 8 minutes

ab216323 was shown to react with CD133 in western blot. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab216323 overnight at 4°C at a 1 in 1000 dilution. Blots were incubated with HRP conjugated Goat anti-Rabbit (H+L) secondary antibody at 1/5000 for 1 hour at room temperature before development with ECL reagent and imaging.

Immunohistochemical analysis of paraffin-embedded human pancreas tissue labeling CD133 with ab216323 at 1/1000 dilution, followed by a Goat Anti-Rabbit IgG H&L (HRP), ready to use. Apical/endoluminal staining on ducts of human pancreas, and negative on Langerhans cells of the islets (PMID: 18261235) is observed. Counter stained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a Goat Anti-Rabbit IgG H&L (HRP), ready to use.
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Flow cytometric analysis of HEK-293T (human embryonic kidney epithelial cell)(Left) / Caco-2 (human colorectal adenocarcinoma epithelial cell)(Right) cell line labeling CD133 with ab216323 at 1/100 (Red) compared with Isotype Control Rabbit monoclonal IgG (ab172730) (BLack) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

Negative control: 293T (PMID 20167130). Total viable cells were gated for the FC image.

All lanes : Anti-CD133 antibody [EPR20980-104] (ab216323) 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

Lysates/proteins at 20 µg per lane.

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

Developed using the ECL technique.

Predicted band size: 97 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking: 5% NFDM/TBST.

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

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

All lanes : Anti-CD133 antibody [EPR20980-104] (ab216323) at 1/1000 dilution

Lane 1 : NCCIT (human pluripotent embryonic carcinoma epithelial cell), whole cell lysate
Lane 2 : Human fetal kidney

Lysates/proteins at 10 µg per lane.

Secondary
Lane 1 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution
Lane 2 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Developed using the ECL technique.

Predicted band size: 97 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?

Blocking: 5% NFDM/TBST.

Exposure time:

Lane 1: 30 seconds

Lane 2: 1 minute

Immunohistochemical analysis of paraffin-embedded human mammary gland tissue labeling CD133 with ab216323 at 1/1000 dilution, followed by a Goat Anti-Rabbit IgG H&L (HRP), ready to use. Apical/endoluminal staining on ducts of human mammary gland (PMID: 18261235) is observed. Counter stained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a Goat Anti-Rabbit IgG H&L (HRP), ready to use.
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling CD133 with ab216323 at 1/1000 dilution, followed by a Goat Anti-Rabbit IgG H&L (HRP), ready to use. Membranous staining on parietal layer of Bowman’s capsule of human kidney (PMID: 19092120) is observed. Counter stained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a Goat Anti-Rabbit IgG H&L (HRP), ready to use.
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

CD133 was immunoprecipitated from 0.35 mg Caco-2 (human colorectal adenocarcinoma epithelial cell) whole cell lysate with ab216323 at 1/70 dilution. Western blot was performed from the immunoprecipitate using ab216323 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1,000 dilution
Lane 1: Caco-2 (human colorectal adenocarcinoma epithelial cell) whole cell lysate 10 μg (Input).
Lane 2: ab216323 IP in Caco-2 whole cell lysate (+).
Lane 3: Rabbit monoclonal IgG (ab172730) instead of  ab216323 in Caco-2 whole cell lysate (-).
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 1 second.