Anti-SIRP alpha antibody [EPR16264] - BSA and Azide free

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-SIRP alpha antibody [EPR16264] - BSA and Azide free (AB271957)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab191419).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SIRP alpha antibody [EPR16264] - BSA and Azide free (AB271957)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab191419).

• WB

Lab

Western blot - Anti-SIRP alpha antibody [EPR16264] - BSA and Azide free (AB271957)

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] (<a href='/en-us/products/primary-antibodies/sirp-alpha-antibody-epr16264-ab191419'>ab191419</a>) 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] - BSA and Azide free (AB271957)

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)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab191419).

All lanes:

Western blot - Anti-SIRP alpha antibody [EPR16264] (<a href='/en-us/products/primary-antibodies/sirp-alpha-antibody-epr16264-ab191419'>ab191419</a>) 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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