Anti-beta 2 Microglobulin antibody [EPR21752-214]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta 2 Microglobulin antibody [EPR21752-214] (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).

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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-beta 2 Microglobulin antibody [EPR21752-214] (AB218230)

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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-beta 2 Microglobulin antibody [EPR21752-214] (AB218230)

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta 2 Microglobulin antibody [EPR21752-214] (AB218230)

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).

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

• IP

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Immunoprecipitation - Anti-beta 2 Microglobulin antibody [EPR21752-214] (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.

All lanes:

Immunoprecipitation - Anti-beta 2 Microglobulin antibody [EPR21752-214] (ab218230)

Predicted band size: 14 kDa

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• WB

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Western blot - Anti-beta 2 Microglobulin antibody [EPR21752-214] (AB218230)

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

Exposure times : Lane 1 : 6 seconds; Lane 2-3 : 37 seconds : Lane 4-7 : 48 seconds; Lane 8-11 : 3 minutes.

Lanes 4-11 : This blot was developed using a higher sensitive ECL substrate.

All lanes:

Western blot - Anti-beta 2 Microglobulin antibody [EPR21752-214] (ab218230) at 1/1000 dilution

Lane 1:

HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg

Lane 2:

Jurkat (human T cell leukemia T lymphocyte), whole cell lysate at 10 µg

Lane 3:

HepG2 (human liver hepatocellular carcinoma cell line) cell lysate at 10 µg

Lane 4:

Mouse serum at 10 µg

Lane 5:

Mouse plasma at 10 µg

Lane 6:

Rat serum at 10 µg

Lane 7:

Rat plasma at 10 µg

Lane 8:

Human plasma at 10 µg

Lane 9:

Human skin lysate at 10 µg

Lane 10:

Human kidney lysate at 10 µg

Lane 11:

Human liver lysate at 10 µg

Secondary

Lanes 1 - 3:

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

Lanes 4 - 11:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 14 kDa

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• WB

Lab

Western blot - Anti-beta 2 Microglobulin antibody [EPR21752-214] (AB218230)

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.

All lanes:

Western blot - Anti-beta 2 Microglobulin antibody [EPR21752-214] (ab218230) at 1/1000 dilution

Lane 1:

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

Lane 2:

B2M knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human B2M (beta 2 Microglobulin) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-b2m-beta-2-microglobulin-knockout-hek-293t-cell-line-ab266828'>ab266828</a>)

Lane 3:

Wild-type A431 cell lysate at 20 µg

Lane 4:

B2M knockout A431 cell lysate 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 [EPR21752-214] (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.

All lanes:

Western blot - Anti-beta 2 Microglobulin antibody [EPR21752-214] (ab218230) at 1/500 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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