Recombinant Anti-beta 2 Microglobulin antibody [EPR21752-214] - BSA and Azide free (ab237032)

False colour image of Western blot: Anti-beta 2 Microglobulin antibody [EPR21752-214] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab218230 was shown to bind specifically to beta 2 Microglobulin. A band was observed at 12 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in B2M knockout cell line ab266828 (knockout cell lysate ab256845). To generate this image, wild-type and B2M knockout HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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.

This data was developed using the same antibody clone in a different buffer formulation (ab218230).

Lanes 1-4: Merged signal (red and green). Green - ab218230 observed at 14 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab218230 Anti-beta 2 Microglobulin antibody [EPR21752-214]  was shown to specifically react with beta 2 Microglobulin in wild-type HepG2 cells. Loss of signal was observed when knockout cell line ab262325 (knockout cell lysate ab256846) was used. Wild-type and beta 2 Microglobulin knockout samples were subjected to SDS-PAGE. ab218230 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling beta 2 Microglobulin with ab218230 at 1/4000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody. Positive staining on endothelial cells of human kidney is observed. Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab218230).

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized  HepG2 (human liver hepatocellular carcinoma cell line) cell line labeling  beta 2 Microglobulin with ab218230 at 1/500 dilution  (red) compared with a Rabbit monoclonal IgG - Isotype control  (ab172730) (black) and an unlabeled control cells incubated with secondary antibody only) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody. 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab218230). 

Beta 2 Microglobulin was immunoprecipitated from 0.35 mg HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab218230 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab218230 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1: HeLa whole cell lysate 10 µg (Input).
Lane 2: ab218230 IP in HeLa whole cell lysate (+).
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab218230 in HeLa whole cell lysate (-).

Blocking/Dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 minutes.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab218230).

Immunohistochemical analysis of paraffin-embedded human spleen tissue labeling beta 2 Microglobulin with ab218230 at 1/4000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody. Positive staining on endothelial cells of human spleen is observed. Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab218230).

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized  HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling  beta 2 Microglobulin with ab218230 at 1/50 dilution  (red) compared with a Rabbit monoclonal IgG - Isotype control  (ab172730) (black) and an unlabeled control (cells incubated with secondary antibody only) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody. 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab218230).