All lanes : Anti-HAI-1 antibody [EPR23294-49] (ab272721) at 1/1000 dilution

Lane 1 : Human placenta tissue lysate
Lane 2 : T47D (human ductal breast epithelial tumor epithelial cell), whole cell lysate
Lane 3 : Daudi (human Burkitts lymphoma lymphoblast), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 58 kDa
Observed band size: 56,66 kDa why is the actual band size different from the predicted?

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

This blot was developed using a higher sensitivity ECL substrate.

Low expression cell line: Daudi (PMID:24070417).

The molecular weight observed is consistent with what has been described in the literature (PMID:11013244 ).

Exposure time: 3 minutes.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized T-47D cells labelling HAI-1 with ab272721 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 (2µg/ml) dilution (Green). Confocal image showing membranous staining in T-47D cell line.

Negative control: Daudi (PMID: 24070417).

ab195889 anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5µg/ml) dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 (2µg/ml) dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Daudi (Human Burkitt's lymphoma lymphoblast) (Left panel) / T-47D (Human ductal breast epithelial tumor epithelial cell) (Right panel) cells labelling HAI-1 with ab272721 at 1/600 dilution (0.1µg) (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: Daudi (PMID: 24070417).

All lanes : Anti-HAI-1 antibody [EPR23294-49] (ab272721) at 1/1000 dilution

Lane 1 : Mouse large intestine tissue lysate
Lane 2 : Mouse small intestine tissue lysate
Lane 3 : Mouse placenta tissue lysate
Lane 4 : Mouse stomach tissue lysate
Lane 5 : Mouse liver tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 58 kDa
Observed band size: 56,66 kDa why is the actual band size different from the predicted?

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

Low expression tissue: liver (PMID:21840293).

The molecular weight observed is consistent with what has been described in the literature (PMID:11013244).

Exposure time: 3 minutes.

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling HAI-1 with ab272721 at 1/2000 followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on germ cells of mouse testis (PMID: 16740973). The section was incubated with ab272721 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 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.

HAI-1 was immunoprecipitated from mouse placenta tissue lysate with ab272721 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab272721 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Mouse placenta tissue lysate 10µg.

Lane 2: ab272721 IP in mouse placenta tissue lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab272721 in mouse placenta tissue lysate.

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

Exposure time: 3 minutes.

This blot was developed using a higher sensitivity ECL substrate.

All lanes : Anti-HAI-1 antibody [EPR23294-49] (ab272721) at 1/1000 dilution

Lane 1 : Rat placenta tissue lysate
Lane 2 : Rat liver tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 58 kDa
Observed band size: 66 kDa why is the actual band size different from the predicted?

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

Low expression tissue: liver (PMID:21840293).

Exposure time: 3 minutes.

Immunohistochemical analysis of paraffin-embedded mouse stomach tissue labeling HAI-1 with ab272721 at 1/2000 followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse stomach. The section was incubated with ab272721 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 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.