Anti-SIRP alpha antibody [EPR16264]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-SIRP alpha antibody [EPR16264] (AB191419)

Intracellular flow cytometric analysis of THP1 cells (2% paraformaldehyde-fixed) labeling SIRP alpha with ab191419 at 1/50 dilution (red) or a Rabbit monoclonal IgG (negative) (green), followed by Goat anti rabbit IgG (FITC) secondary at 1/150 dilution

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SIRP alpha antibody [EPR16264] (AB191419)

Immunocytochemistry/ Immunofluorescence analysis of C6 (rat glial tumor glial cell) labeling SIRP alpha with ab191419 at 1/500. ab150077 Alexa Fluor^® 488 Goat anti-Rabbit at 1/1000 was used as the secondary antibody. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. DAPI was used to stain nuclei blue.

Confocal image showing membranous staining on C6 cell line.

• WB

Lab

Western blot - Anti-SIRP alpha antibody [EPR16264] (AB191419)

Western blot : Anti-SIRPA antibody [EPR16264] (ab191419) staining at 1/1500 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab191419 was shown to bind specifically to SIRPA. A band was observed at 75-98 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 [EPR16264] (ab191419) at 1/1500 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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• WB

Lab

Western blot - Anti-SIRP alpha antibody [EPR16264] (AB191419)

Lanes 1 - 4 : Merged signal (red and green). Green - ab191419 observed at 55 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab191419 was shown to recognize SIRP alpha in wild-type HAP1 cells as signal was lost at the expected MW in SIRPA knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and SIRPA knockout samples were subjected to SDS-PAGE. ab191419 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 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.

All lanes:

Western blot - Anti-SIRP alpha antibody [EPR16264] (ab191419) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

SIRPA knockout HAP1 whole cell lysate at 20 µg

Lane 3:

THP1 whole cell lysate at 20 µg

Lane 4:

Jurkat whole cell lysate (negative control) at 20 µg

Predicted band size: 55 kDa

Observed band size: 55 kDa

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• WB

Lab

Western blot - Anti-SIRP alpha antibody [EPR16264] (AB191419)

False colour image of Western blot : Anti-SIRP alpha antibody [EPR16264] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab191419 was shown to bind specifically to SIRP alpha. A band was observed at 100-140 kDa (mouse SIRPA, isoform 1) & 40-50 kDa (mouse SIRPA, isoform 2), in wild-type RAW 264.7 cell lysates (band observed at 70-100 kDa in THP-1 is Human SIRPA) with no signal observed at this size in SIRPA knockout cell line ab281618 (knockout cell lysate ab282969). To generate this image, wild-type and SIRPA knockout RAW 264.7 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Glycosylation level (~65-120 kDa) of SIRPα is different in various tissues (PMID : 18051954).

observed band : 100-140 kDa (mouse SIRPA, isoform 1) & 40-50 kDa (mouse SIRPA, isoform 2)

All lanes:

Western blot - Anti-SIRP alpha antibody [EPR16264] (ab191419) at 1/1000 dilution

Lane 1:

Wild-type RAW 264.7 cell lysate at 20 µg

Lane 2:

SIRPA knockout RAW 264.7 cell lysate at 20 µg

Lane 2:

Western blot - Mouse SIRPA knockout RAW 264.7 cell line (<a href='/en-us/products/cell-lines/mouse-sirpa-knockout-raw-2647-cell-line-ab281618'>ab281618</a>)

Lane 3:

THP-1 cell lysate at 20 µg

Lane 4:

MCF7 cell lysate at 20 µg

Predicted band size: 55 kDa

Observed band size: 100-140 kDa

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• WB

Supplier Data

Western blot - Anti-SIRP alpha antibody [EPR16264] (AB191419)

All lanes:

Western blot - Anti-SIRP alpha antibody [EPR16264] (ab191419) at 1/1000 dilution

Lane 1:

C6 cell lysate at 10 µg

Lane 2:

PC12 cell lysate at 10 µg

Lane 3:

NIH 3T3 cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 55 kDa

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• WB

Supplier Data

Western blot - Anti-SIRP alpha antibody [EPR16264] (AB191419)

All lanes:

Western blot - Anti-SIRP alpha antibody [EPR16264] (ab191419) at 1/5000 dilution

Lane 1:

THP1 cell lysate at 20 µg

Lane 2:

SW480 cell lysate at 20 µg

Lane 3:

Human fetal brain lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 55 kDa

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