Anti-beta 2 Microglobulin antibody [EP2978Y]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-beta 2 Microglobulin antibody [EP2978Y] (AB75853)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-beta 2 Microglobulin antibody [EP2978Y] (AB75853)

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).

• IP

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Immunoprecipitation - Anti-beta 2 Microglobulin antibody [EP2978Y] (AB75853)

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.

All lanes:

Immunoprecipitation - Anti-beta 2 Microglobulin antibody [EP2978Y] (ab75853)

Predicted band size: 14 kDa

Observed band size: 14 kDa

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• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-beta 2 Microglobulin antibody [EP2978Y] (AB75853)

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.

• WB

Lab

Western blot - Anti-beta 2 Microglobulin antibody [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.

All lanes:

Western blot - Anti-beta 2 Microglobulin antibody [EP2978Y] (ab75853) at 1/5000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

B2M knockout A549 cell lysate at 20 µg

Lane 3:

Wild-type HEK-293T ab255553 cell lysate at 20 µg

Lane 4:

B2M knockout HEK-293T <a href='/en-us/products/cell-lines/human-b2m-beta-2-microglobulin-knockout-hek-293t-cell-line-ab266828'>ab266828</a> cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

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• WB

Lab

Western blot - Anti-beta 2 Microglobulin antibody [EP2978Y] (AB75853)

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.

All lanes:

Western blot - Anti-beta 2 Microglobulin antibody [EP2978Y] (ab75853) at 1/1000 dilution

Lane 1:

Wild-type HepG2 cell lysate at 20 µg

Lane 2:

B2M knockout HepG2 cell lysate at 20 µg

Lane 2:

Western blot - Human B2M (beta 2 Microglobulin) knockout Hep G2 cell line (<a href='/en-us/products/cell-lines/human-b2m-beta-2-microglobulin-knockout-hep-g2-cell-line-ab262325'>ab262325</a>)

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 14 kDa

Observed band size: 14 kDa

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• WB

Lab

Western blot - Anti-beta 2 Microglobulin antibody [EP2978Y] (AB75853)

Blocking and dilution buffer : 5% NFDM /TBST.

All lanes:

Western blot - Anti-beta 2 Microglobulin antibody [EP2978Y] (ab75853) at 1/5000 dilution

Lane 1:

U937 whole cell lysate at 20 µg

Lane 2:

HeLa 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 1/20000 dilution

Predicted band size: 14 kDa

Observed band size: 14 kDa

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• WB

Lab

Western blot - Anti-beta 2 Microglobulin antibody [EP2978Y] (AB75853)

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.

All lanes:

Western blot - Anti-beta 2 Microglobulin antibody [EP2978Y] (ab75853) at 1/5000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human B2M (beta 2 Microglobulin) knockout HEK-293T cell lysate (<a href='/en-us/products/cell-lysates/human-b2m-beta-2-microglobulin-knockout-hek-293t-cell-lysate-ab256845'>ab256845</a>) at 20 µg

Lane 3:

Wild-type A431 cell lysate at 20 µg

Lane 4:

Western blot - Human B2M (beta 2 Microglobulin) knockout A-431 cell lysate (<a href='/en-us/products/cell-lysates/human-b2m-beta-2-microglobulin-knockout-a-431-cell-lysate-ab261702'>ab261702</a>) at 20 µg

Predicted band size: 14 kDa

Observed band size: 12 kDa

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• WB

Lab

Western blot - Anti-beta 2 Microglobulin antibody [EP2978Y] (AB75853)

Blocking and dilution buffer : 5% NFDM /TBST.

All lanes:

Western blot - Anti-beta 2 Microglobulin antibody [EP2978Y] (ab75853) at 1/5000 dilution

Lane 1:

Mouse kidney tissue lysate at 20 µg

Lane 2:

Rat kidney tissue 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 1/20000 dilution

Predicted band size: 14 kDa

Observed band size: 14 kDa

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• WB

Unknown

Western blot - Anti-beta 2 Microglobulin antibody [EP2978Y] (AB75853)

All lanes:

Western blot - Anti-beta 2 Microglobulin antibody [EP2978Y] (ab75853) at 1/200000 dilution

Lane 1:

Raji cell lysate at 10 µg

Lane 2:

U937 cell lysate at 10 µg

Lane 3:

HeLa cell lysate at 10 µg

Lane 4:

Human bone marrow lysate at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 14 kDa

Observed band size: 14 kDa

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