Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: Scavenging Receptor SR-BI knockout HAP1 whole cell lysate (20 µg)
Lane 3: HepG2 whole cell lysate (20 µg)
Lane 4: Human liver whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab52629 observed at 80 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab52629 was shown to specifically react with Scavenging Receptor SR-BI in wild-type HAP1 cells as signal was lost in Scavenging Receptor SR-BI knockout cells. Wild-type and Scavenging Receptor SR-BI knockout samples were subjected to SDS-PAGE. Ab52629 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/2000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Scavenging Receptor SR-BI antibody [EP1556Y] (ab52629) at 1/1000 dilution (purified)

Lane 1 : Mouse liver lysate
Lane 2 : Rat liver lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Anti-rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Observed band size: 80 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

All lanes : Anti-Scavenging Receptor SR-BI antibody [EP1556Y] (ab52629) at 1/1000 dilution (purified)

Lane 1 : Human fetal liver lysate
Lane 2 : HepG2 lysate
Lane 3 : PC-3 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Anti-rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Observed band size: 80 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

Immunohistochemical staining of paraffin embedded human liver with purified ab52629 at a working dilution of 1/500. The secondary antibody used is ab97051, a goat anti-rabbit IgG (H&L) at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Anti-Scavenging Receptor SR-BI antibody [EP1556Y] (ab52629) at 1/2000 dilution (unpurified) + Mouse liver lysate at 10 µg

Secondary
goat anti-rabbit HRP at 1/2000 dilution

Observed band size: 80 kDa why is the actual band size different from the predicted?

THP1 cells were incubated at 37ºC for 40h with vehicle control (0 µM) and different concentrations of sodium salicylate (ab120746). Decreased expression of scavenging receptor SR-BI in THP1 cells correlates with an increase in sodium salicylate concentration, as described in literature.

Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 10 µg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with unpurified ab52629 at 1/2000 dilution and ab8227 at 1 µg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.