重组Anti-MERTK抗体[Y323] - BSA and Azide free (ab271851)

This data was developed using the same antibody clone in a different buffer formulation (ab52968).

Western blot: Anti-MERTK antibody [Y323] (ab52968) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab52968 was shown to bind specifically to MERTK. A band was observed at 160 kDa in wild-type A549 cell lysates with no signal observed at this size in MERTK knockout cell line. To generate this image, wild-type and MERTK knockout A549 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52968).

Lanes 1 - 2: Merged signal (red and green). Green - ab52968 observed at 110 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab52968 was shown to specifically react with MERTK in wild-type HAP1 cells as signal was lost in MERTK knockout cells. Wild-type and MERTK knockout samples were subjected to SDS-PAGE. The membrane was blocked with 0. Ab52968 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/500 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52968).

ab52968 used at a 1/200 dilution, 18 hours at 4°C, 5% BSA in Tween PBS.

Positive cell line: A172

Negative cell lines: CV1 and HeLa.

Secondary used is a goat anti-rabbit polyclonal-HRP at 1/10000 dilution.

Blocked with 5% milk for 1 hour at RT.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52968).

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

Lane 1: ab52968 (purified) at 1/20 immunoprecipitating MERTK in HEK-293 (Human epithelial cell line from embryonic kidney) cell lysate.

Lane 2 - PBS.

For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500).
Blocking/Dilution buffer and concentration: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labeling MERTK with purified ab52968 at 1/500.

Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Counterstained with hematoxylin.

Negative control using PBS instead of primary antibody (Inset).

This data was developed using the same antibody clone in a different buffer formulation (ab52968).

IHC image of MERTK staining in a section of frozen normal human spleen performed on a Leica BOND^TM system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab52968, 1/1000 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52968).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lymphoma tissue labeling MERTK with unpurified ab52968 at a 1/50 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.