Anti-Apo-H antibody [EPR23087-228] (ab267464) at 1/1000 dilution + Human plasma at 20 µg

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

Predicted band size: 38 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 180 seconds.

The molecular weight observed is consistent with what has been described in the literature (PMID: 30894461).

 

All lanes : Anti-Apo-H antibody [EPR23087-228] (ab267464) at 1/1000 dilution

Lane 1 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : Human liver 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: 38 kDa
Observed band size: 50,60 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 147 seconds.

The molecular weight observed is consistent with what has been described in the literature (PMID: 30894461)

Negative control: Hela (PMID: 14984368).

Apo-H was immunoprecipitated from 0.35 mg HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate with ab267464 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab267464 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

Lane 1: HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate 10ug

Lane 2: ab267464 IP in HepG2 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab267464 in HepG2 whole cell lysate

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

Exposure time: 30 seconds

 

Immunohistochemical analysis of paraffin-embedded Human hepatocellular carcinoma tissue labeling Apo-H with ab267464 at 1/500 dilution (0.94 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on plasma in human hepatocellular carcinoma. The section was incubated with ab267464 for 15 mins at RT. The immunostaining staining 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 solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Apo-H with ab267464 at 1/500 dilution (0.94 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on plasma in human colon. The section was incubated with ab267464 for 15 mins at RT. The immunostaining staining 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 solution 2) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labelling Apo-H with ab267464 at 1/100 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in HepG2 cell line, and no staining in Hela cells. Negative control: Hela (PMID: 14984368). ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell, Left) / HepG2 (human hepatocellular carcinoma epithelial cell, Right) cells labelling Apo-H with ab267464 at 1/500 dilution (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). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control: HeLa (PMID: 14984368).