Anti-BAFF antibody [EPR22238]

• Flow Cyt

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Flow Cytometry - Anti-BAFF antibody [EPR22238] (AB224710)

Flow cytometric analysis of Daudi (human Burkitt's lymphoma cell line; left panel) and HL-60 (human promyelocytic leukemia cell line ;right panel) cell lines labeling BAFF with ab224710 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control : Daudi. (PMID : 20951740)

Gated on viable cells.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-BAFF antibody [EPR22238] (AB224710)

Immunofluorescent analysis of 100% methanol-fixed U937 (human histiocytic lymphoma cell line) cells labeling BAFF with ab224710 at 1/100 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on U937 cells.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Confocal image showing membranous staining in U-937 cells.

We don't recommend PFA fixation, since the result shows some background staining in the nuclear.

Negative control : HEK-293（PMID : 17393395.

• IP

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Immunoprecipitation - Anti-BAFF antibody [EPR22238] (AB224710)

BAFF was immunoprecipitated from 0.35 mg of U937 (human histiocytic lymphoma cell line) whole cell lysate with ab224710 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab224710 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1 : U937 whole cell lysate 10 μg (Input).

Lane 2 : ab224710 IP in U937 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab224710 in U937 whole cell lysate.

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

Exposure time : 20 seconds.

All lanes:

Immunoprecipitation - Anti-BAFF antibody [EPR22238] (ab224710)

Predicted band size: 31 kDa

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• WB

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Western blot - Anti-BAFF antibody [EPR22238] (AB224710)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-BAFF antibody [EPR22238] (ab224710) at 1/1000 dilution

Lane 1:

U937 (human histiocytic lymphoma cell line) whole cell lysate at 20 µg

Lane 2:

HL-60 (human promyelocytic leukemia cell line) whole cell lysate at 20 µg

Lane 3:

HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 4:

Daudi (human Burkitt's lymphoma cell line) 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 1/50000 dilution

Predicted band size: 31 kDa

Observed band size: 30 kDa,32 kDa

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Exposure time: 26s