重组Anti-beta 2 Microglobulin抗体[EP2978Y] (ab75853)

Western blot: Anti-B2M antibody [EP2978Y] (ab75853) staining at 1/5000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab75853 was shown to bind specifically to B2M. A band was observed at 13 kDa in wild-type A549 cell lysates with no signal observed at this size in B2M knockout cell line. To generate this image, wild-type and B2M 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 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

Flow cytometry overlay histogram showing C57 BL/6 mouse splenocytes stained with ab75853 (red line). Cells were fixed and permeabilised with BD Cytofix/Cytoperm™ for 20 min. The cells were incubated in 1x PBS containing 10μg/ml anti CD16/CD32 and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab75853) (1x 10⁶ in 100μl at 0.2 μg/ml (1/11300)) for 30min on ice.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min on ice

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

Western blot: Anti-beta 2 Microglobulin antibody [EP2978Y] staining at 1/5000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A]  (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab75853 was shown to bind specifically to beta 2 Microglobulin. A band was observed at 12 kDa in Wild-type A431 and HEK-293T cell lysates with no signal observed at this size in B2M (beta 2 Microglobulin) knockout A-431 cell line ab261893 (knockout cell lysate ab261702) or B2M knockout HEK-293T cell line ab266828 (knockout cell lysate ab256845). To generate this image, wild-type and B2M knockout A431 and HEK-293T cell lysates were analysed.
Nitrocellulose membranes were blocked in fluorescent western blot (TBS-based) blocking solution in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed in TBS-T, incubated with secondary antibodies Goat anti-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20000 dilution for 1 h at room temperature, washed again then imaged.

Lanes 1-4: Merged signal (red and green). Green - ab75853 observed at 14 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab75853 Anti-beta 2 Microglobulin antibody [EP2978Y] 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. ab75853 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 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.

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labelling beta 2 Microglobulin with purified ab75853 at 1/20 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies. 

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling beta 2 Microglobulin with purified ab75853 at 1/100. Cells were fixed with 100% methanol and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

Blocking and dilution buffer: 5% NFDM /TBST.

Blocking and dilution buffer: 5% NFDM /TBST.

ab75853 (purified) at 1/20 immunoprecipitating beta 2 Microglobulin in Raji whole cell lysate.

Lane 1 (input): Raji whole cell lysate (10µg)

Lane 2 (+): ab75853 + Raji whole cell lysate (10µg).

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab75853 in Raji whole cell lysate.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.