Anti-SHIP-1 antibody [EP378Y]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SHIP-1 antibody [EP378Y] (AB45142)

Immunocytochemistry/Immunofluorescence analysis of Raji cells labelling SHIP-1 with purified ab45142 at 1/150. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077 an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291 a mouse anti-tubulin (1/1000) and ab150120 an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1 : primary antibody (1/150) and secondary antibody ab150120 an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2 : ab7291 (1/1000) and secondary antibody ab150077 an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-SHIP-1 antibody [EP378Y] (AB45142)

Intracellular Flow Cytometry analysis of Raji cells labelling SHIP-1 with purified ab45142 at 1/50 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SHIP-1 antibody [EP378Y] (AB45142)

Immunocytochemistry/Immunofluorescence analysis of Jurkat cells labelling SHIP-1 with purified ab45142 at 1/150. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077 an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291 a mouse anti-tubulin (1/1000) and ab150120 an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1 : primary antibody (1/150) and secondary antibody ab150120 an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2 : ab7291 (1/1000) and secondary antibody ab150077 an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-SHIP-1 antibody [EP378Y] (AB45142)

Flow cytometry overlay histogram showing left Raji positive cells and right negative MCF7 stained with ab45142 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10μg/ml human IgG and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab45142) (1x 106 in 100μl at 0.008μg/ml (1/275000)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SHIP-1 antibody [EP378Y] (AB45142)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human spleen tissue labelling SHIP-1 with purified ab45142 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). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SHIP-1 antibody [EP378Y] (AB45142)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human spleen tissue labelling SHIP-1 with unpurified ab45142 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SHIP-1 antibody [EP378Y] (AB45142)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse spleen tissue labelling SHIP-1 with purified ab45142 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). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• WB

Lab

Western blot - Anti-SHIP-1 antibody [EP378Y] (AB45142)

ab45142 was shown to react with INPP5D in wild-type iPSC-derived microglia cells in Western blot with loss of signal observed in a INPP5D knockout cell line. Wild-type iPSC-derived microglia and INPP5D knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab45142 overnight at 4 °C at a 1/500 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-SHIP-1 antibody [EP378Y] (ab45142) at 1/500 dilution

Lane 1:

Wild-type iPSC-derived microglia lysate at 10 µg

Lane 2:

INPP5D knock-out iPSC-derived microglia lysate at 10 µg

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• IP

Lab

Immunoprecipitation - Anti-SHIP-1 antibody [EP378Y] (AB45142)

ab45142 (purified) at 1/20 immunoprecipitating SHIP-1 in Daudi whole cell lysate.

Lane 1 (input) : Daudi whole cell lysate (10μg)

Lane 2 (+) : ab45142 + Daudi whole cell lysate (10μg).

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab45142 in Daudi whole cell lysate.

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 buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-SHIP-1 antibody [EP378Y] (ab45142)

Predicted band size: 133 kDa

Observed band size: 140 kDa

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• WB

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Western blot - Anti-SHIP-1 antibody [EP378Y] (AB45142)

All lanes:

Western blot - Anti-SHIP-1 antibody [EP378Y] (ab45142) at 1/50000 dilution

All lanes:

Daudi cell lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 133 kDa

Observed band size: 140 kDa

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• WB

Lab

Western blot - Anti-SHIP-1 antibody [EP378Y] (AB45142)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-SHIP-1 antibody [EP378Y] (ab45142) at 1/1000 dilution

All lanes:

KM3 cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 133 kDa

Observed band size: 140 kDa

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• WB

Lab

Western blot - Anti-SHIP-1 antibody [EP378Y] (AB45142)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-SHIP-1 antibody [EP378Y] (ab45142) at 1/5000 dilution

Lane 1:

Daudi cell lysate at 20 µg

Lane 2:

Ramos cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 133 kDa

Observed band size: 140 kDa

false