All lanes : Anti-MERTK antibody [Y323] (ab52968) at 1/500 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : MERTK knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

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.

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.

See Abreview

All lanes : Anti-MERTK antibody [Y323] (ab52968) at 1/2000 dilution (purified)

Lane 1 : HEK-293 (Human epithelial cell line from embryonic kidney) cell lysate
Lane 2 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG at 1/1000 dilution

Observed band size: 140-180 kDa why is the actual band size different from the predicted?

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).

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.

Anti-MERTK antibody [Y323] (ab52968) at 1/1000 dilution (unpurified) + HEK-293 (Human epithelial cell line from embryonic kidney) cell lysate at 10 µg

Secondary
HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Observed band size: 180 kDa why is the actual band size different from the predicted?

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.