Recombinant Anti-Scavenging Receptor SRB2 antibody [EPR12081] - C-terminal (ab196651)

All lanes : Anti-Scavenging Receptor SRB2 antibody [EPR12081] - C-terminal (ab196651) at 1/20000 dilution

Lane 1 : Wild-type MCF7 cell lysate
Lane 2 : SCARB2 knockout MCF7 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 54 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

False colour image of Western blot: Anti-Scavenging Receptor SRB2 antibody [EPR12081] - C-terminal staining at 1/20000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab196651 was shown to bind specifically to Scavenging Receptor SRB2. A band was observed at 80 kDa in wild-type MCF7 cell lysates with no signal observed at this size in SCARB2 knockout cell line ab274952 (knockout cell lysate ab275010). To generate this image, wild-type and SCARB2 knockout MCF7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes : Anti-Scavenging Receptor SRB2 antibody [EPR12081] - C-terminal (ab196651) at 1/20000 dilution

Lane 1 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lane 2 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 54 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

The observed MW is consistent with what has been described in the literature (PMID: 21389126, PMID: 22272359 and PMID: 23635510).

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Scavenging Receptor SRB2 with ab196651 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Cytoplasm staining on human liver tissue is observed. Counter stained with Hematoxylin. Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Anti-Scavenging Receptor SRB2 antibody [EPR12081] - C-terminal (ab196651) at 1/20000 dilution + Human fetal kidney lysate at 10 µg

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

Predicted band size: 54 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

The observed MW is consistent with what has been described in the literature (PMID: 21389126, PMID: 22272359 and PMID: 23635510).

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Scavenging Receptor SRB2 with ab196651 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Cytoplasm staining on mouse liver tissue is observed. Counter stained with Hematoxylin. Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Negative control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Intracellular flow cytometric analysis of 2% paraformaldehyde-fixed MCF7 (Human breast adenocarcinoma cell line) cells labeling Scavenging Receptor SRB2 with ab196651 at 1/150 dilution (red) compared with a rabbit monoclonal IgG isotype control (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.