BCMA was immunoprecipitated from 0.35 mg of U266B1 (human multiple myeloma B lymphocyte cell line) whole cell lysate with ab253242 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab253242 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/1000 dilution.

Lane 1: U266B1 whole cell lysate 10 μg (Input).
Lane 2: ab253242 IP in U266B1 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab253242 in U266B1 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 10 seconds.

Flow cytometric analysis of  HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) (left) or U266B1 (human multiple myeloma B lymphocyte cell line) (right) cells labeling BCMA with ab253242 at 1/60 dilution (red) compared with Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled 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.

Gated on viable cells.

Negative control: HEK-293T cells（PMID: 18025285.

 

All lanes : Anti-BCMA antibody [EPR22457-260] (ab253242) at 1/1000 dilution

Lane 1 : U266B1 (human multiple myeloma B lymphocyte cell line) whole cell lysate
Lane 2 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 20, 14 kDa
Observed band size: 19 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and dilution buffer: 5% NFDM/TBST.

BCMA is a glycoprotein.
The molecular weight observed is consistent with what has been described in the literature (PMID: 23776238, 10908663,
8527407)

Negative control: HEK-293T (PMID: 18025285).

 

Anti-BCMA antibody [EPR22457-260] (ab253242) at 1/1000 dilution + Recombinant his-tagged human BCMA protein (ab219697, aa1-54) at 0.02 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 20, 14 kDa

Blocking and dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U288B1 (human multiple myeloma B lymphocyte cell line) or HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells labeling BCMA with ab253242 at 1/50 dilution, followed by a Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).
Confocal image showing cytoplasmic and weak membranous staining in U266B1 cell line (PMID: 8527407) is observed.
Negative control: HEK-293T cells（PMID: 18025285).
The nuclear conter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

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