Anti-SPARC antibody [EPR20121]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-SPARC antibody [EPR20121] (AB207743)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed A-375 (Human malignant melanoma cell line) cells labeling SPARCwith ab207743 at 1/70 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

• IP

Supplier Data

Immunoprecipitation - Anti-SPARC antibody [EPR20121] (AB207743)

SPARC was immunoprecipitated from 0.35 mg of A-375 (Human malignant melanoma cell line) whole cell lysate with ab207743 at 1/40 dilution.

Western blot was performed from the immunoprecipitate using ab207743 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : A-375 whole cell lysate, 10μg (Input).

Lane 2 : ab207743 IP in A-375 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab207743 in A-375 whole cell lysate.

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

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-SPARC antibody [EPR20121] (ab207743)

Predicted band size: 35 kDa

Observed band size: 43 kDa

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• WB

Supplier Data

Western blot - Anti-SPARC antibody [EPR20121] (AB207743)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-SPARC antibody [EPR20121] (ab207743) at 1/1000 dilution

Lane 1:

Human serum at 10 µg

Lane 2:

Human brain lysate at 10 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/4000 dilution

Predicted band size: 35 kDa

Observed band size: 43 kDa

false

Exposure time: 5s

• WB

Supplier Data

Western blot - Anti-SPARC antibody [EPR20121] (AB207743)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 : 30 seconds; Lane 2/3 : 3 minutes.

The observed molecular weight is consistent with what has been described in the literature (PMID : 18442048).

All lanes:

Western blot - Anti-SPARC antibody [EPR20121] (ab207743) at 1/1000 dilution

Lane 1:

SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate at 20 µg

Lane 2:

A-375 (Human malignant melanoma cell line) whole cell lysate at 20 µg

Lane 3:

Human bone giant cell tumor lysate at 20 µg

Secondary

Lanes 1 - 2:

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

Lane 3:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/4000 dilution

Predicted band size: 35 kDa

Observed band size: 43 kDa

false

• WB

Lab

Western blot - Anti-SPARC antibody [EPR20121] (AB207743)

Lanes 1 - 2 : Merged signal (red and green). Green - ab207743 observed at 35 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab207743 was shown to recognize SPARC in wild-type HAP1 cells as signal was lost at the expected MW in SPARC knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and SPARC knockout samples were subjected to SDS-PAGE. ab207743 and ab130007 (Mouse anti-Vinculin 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-SPARC antibody [EPR20121] (ab207743) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

SPARC knockout HAP1 whole cell lysate at 20 µg

Predicted band size: 35 kDa

Observed band size: 35 kDa

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