Anti-Phosphatidylinositide phosphatase SAC2 (phospho S940) antibody [MJF-R31301-10]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Phosphatidylinositide phosphatase SAC2 (phospho S940) antibody [MJF-R31301-10] (AB325150)

Immunohistochemical staining of formalin-fixed and paraffin-embedded rat normal Substantia nigra, labelling INPP5F, phospho Ser940 with ab325150 at 0.25µg/ml, displaying cytoplasmic puncta characteristic of the target (examples are marked by arrows). The procedure was performed using the Leica Biosystems BOND™ RX instrument.

Heat mediated antigen retrieval was performed with EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins at 100°C.

Prior to primary antibody addition, alkaline phosphatase treatment was performed (AP+, bottom) for 60mins at 37°C. For the treatment negative control image (AP-, top), the same treatment was performed, but with the enzyme substituted for distilled water.

Sections were then incubated with ab325150 for 15mins at room temperature, followed by detection with the BOND™ Polymer Refine Detection kit (ready to use, containing secondary antibody, chromogen and hematoxylin).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Phosphatidylinositide phosphatase SAC2 (phospho S940) antibody [MJF-R31301-10] (AB325150)

Immunohistochemical staining of formalin-fixed and paraffin-embedded HEK cell pellets, labelling INPP5F, phospho Ser940 with ab325150 at 0.25µg/ml. The procedure was performed using the Leica Biosystems BOND™ RX instrument.

Heat mediated antigen retrieval was performed with EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins at 100°C.

Prior to primary antibody addition, alkaline phosphatase treatment was performed for 60mins at 37°C (right). For the treatment negative control images (untreated, left) the same treatment was performed, but with the enzyme substituted for distilled water.

Sections were then incubated with ab325150 for 15mins at room temperature, followed by detection with the BOND™ Polymer Refine Detection kit (ready to use, containing secondary antibody, chromogen and hematoxylin).

The control wt cells (A. & B. expected to not express the target) show no staining. The target transiently overexpressing HEK cells untreated (C., expected to strongly express the target), show target staining. Whilst the same cells treated with alkaline phosphatase (D., expected for signal to be lost following alkaline phosphatase treatment), show no staining. The HEK cells transiently overexpressing mutant targets (E. & F., Ser940Asp and G. & H., Ser940Ala, expected to not express the target) all show no staining.

• WB

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Western blot - Anti-Phosphatidylinositide phosphatase SAC2 (phospho S940) antibody [MJF-R31301-10] (AB325150)

False colour image of Western blot : Anti-INPP5F-Ser940 antibody [EPR31301-10] - (ab325150) staining at 1/400 dilution, shown in green; Mouse anti-GAPDH loading control staining, shown in magenta. In Western blot, ab325150 was shown to bind specifically to INPP5F-Ser940. A band was observed at 150 kDa in HEK-293T eGFP INPP5F-wild type cell lysates, with no signal observed at this size in wild-type HEK-293, HEK-293T eGFP INPP5F-Ser940Ala or HEK-293TeGFP INPP5F-Ser940Asp mutant cell lysates. Samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % BSA 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 at 1 : 20000 and Goat anti-mouse IgG H&L 680RD at 1 : 20000.

All lanes:

Western blot - Anti-Phosphatidylinositide phosphatase SAC2 (phospho S940) antibody [MJF-R31301-10] (ab325150) at 1/400 dilution

Lane 1:

HEK-293 wild type whole cell lysate at 20 µg

Lane 2:

HEK-293T eGFP INPP5F-wild type whole cell lysate at 20 µg

Lane 3:

HEK-293T eGFP INPP5F-Ser940Ala whole cell lysate at 20 µg

Lane 4:

HEK-293TeGFP INPP5F-Ser940Asp whole cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat Anti-Rabbit IgG H&L (IRDye® 800CW) at 1/20000 dilution

Lanes 1 - 4:

Goat Anti-Mouse IgG H&L (IRDye® 680RD) at 1/20000 dilution

Observed band size: 150 kDa,36 kDa

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