Anti-SIRP alpha antibody [EPR22930-163]

• Flow Cyt

Lab

Flow Cytometry - Anti-SIRP alpha antibody [EPR22930-163] (AB260039)

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) with ab260039 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left). PBMCs were incubated for 30 mins on ice in 1x PBS containing 10 µg/ml human IgG and 10 % normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody ab260039 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 10⁶ in 100 µl at 0.2 μg/ml (1/2,500 dilution)) for 30 mins on ice. The cells were simultaneously stained with CD14.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 dilution for 30 mins on ice

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on CD14+.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SIRP alpha antibody [EPR22930-163] (AB260039)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of formaldehyde-fixed human lung tissue staining with ab260039 at 1/250 dilution. Sections were cut at 2 µm from lung tissues and mounted on poly-L-lysin coated slides. Samples were incubated with the primary antibody with BSA 5% in PBS for 16 hours at 25°C. Then, the slides were washed in PBS pH 7.4, incubated with secondary antibody Alexa Fluor™ 568 Goat anti-rabbit IgG(H+L) at 1/250 dilution for 30 min at room temperature. Blocking was done using 5% BSA for 20 minutes at 25°C. The second step was performed using primary antibody to CD68 for 1h at room temperature. Then, the slides were washed in PBS pH 7.4, incubated with the appropriate anti mouse secondary antibody 1/250 dilution for 30 min and the DNA was counterstained with Hoechst 33342 at 1µg/ml for 30 min at room temperature. After final washes with PBS, coverslips were mounted on the top of the samples using homemade Mowiol mounting medium.

This image is courtesy of an Abreview submitted by Christos Efstathiou

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SIRP alpha antibody [EPR22930-163] (AB260039)

Immunohistochemical analysis of paraffin-embedded human spleen tissue labeling SIRP alpha with ab260039 at 1/500 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on the human spleen is observed. The section was incubated with ab260039 for 15 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control : Ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SIRP alpha antibody [EPR22930-163] (AB260039)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SK-MEL-28 (human malignant melanoma melanoma) cells labeling SIRP alpha with ab260039 at 1/250 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing membranous staining in SK-MEL-28 cells Negative control : Jurkat (PMID : 17982459) is observed. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control : ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

• Flow Cyt

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Flow Cytometry - Anti-SIRP alpha antibody [EPR22930-163] (AB260039)

Flow cytometric analysis of Jurkat (Human T cell leukemia T lymphocyte, Left) / THP-1 (Human monocytic leukemia monocyte, Right) cells labeling SIRP alpha with ab260039 at 1\600 compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 was used as the secondary antibody.

Negative control : Jurkat.

Gated on viable cells.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SIRP alpha antibody [EPR22930-163] (AB260039)

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling SIRP alpha with ab260039 at 1/500 followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on the human cerebrum (PMID : 9062191) is observed. The section was incubated with ab260039 for 15 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control : Ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• WB

Lab

Western blot - Anti-SIRP alpha antibody [EPR22930-163] (AB260039)

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

There are many forms of SIRP alpha because of different glycosylation and phosphotyrosine modification.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 9712903).

Negative control : Jurkat (PMID : 17982459).

Exposure time : 3 minutes

All lanes:

Western blot - Anti-SIRP alpha antibody [EPR22930-163] (ab260039) at 1/1000 dilution

Lane 1:

Human brain lysate at 20 µg

Lane 2:

U-937 (human histiocytic lymphoma monocyte) whole cell lysate at 20 µg

Lane 3:

SK-MEL-28 (human malignant melanoma) whole cell lysate at 20 µg

Lane 4:

SK-MEL-2 (human skin malignant melanoma) whole cell lysate at 20 µg

Lane 5:

Jurkat (human T-cell leukemia lymphocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 55 kDa

Observed band size: 85 kDa

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• WB

Lab

Western blot - Anti-SIRP alpha antibody [EPR22930-163] (AB260039)

Western blot : Anti-SIRPA antibody [EPR22930-163] (ab260039) 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, ab260039 was shown to bind specifically to SIRPA. A band was observed at 75-85 kDa in wild-type A549 cell lysates with no signal observed at this size in SIRPA knockout cell line. To generate this image, wild-type and SIRPA 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.

All lanes:

Western blot - Anti-SIRP alpha antibody [EPR22930-163] (ab260039) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

SIRPA knockout A549 cell lysate at 20 µg

Lane 3:

THP-1 cell lysate at 20 µg

Lane 4:

SW480 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

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