Anti-beta 2 Microglobulin antibody [EPR21752-214]

8 product images

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  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 Anti-GAPDH antibody [6C5] - 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 Human B2M (beta 2 Microglobulin) knockout Hep G2 cell line ab262325 (knockout cell lysate Human B2M (beta 2 Microglobulin) knockout Hep G2 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 (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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (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 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/10000 dilution

  Predicted band size: 14 kDa

  Observed band size: 14 kDa

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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.

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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) (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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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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  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] (Anti-alpha Tubulin antibody [DM1A] - Loading Control 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 Human B2M (beta 2 Microglobulin) knockout HEK-293T cell line ab266828 (knockout cell lysate Human B2M (beta 2 Microglobulin) knockout HEK-293T 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed 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 3: Wild-type A431 cell lysate at 20 µg

  Lane 4: B2M knockout A431 cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 14 kDa

  Observed band size: 12 kDa

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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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (black) and an unlabeled control cells incubatedwith secondary antibody only) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077), at 1/2000 dilution was used as the secondary antibody.
  

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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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Lanes 10, 11, 4, 5, 6, 7, 8 and 9: Western blot - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/1000 dilution

  Predicted band size: 14 kDa

• 

  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 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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (black) and an unlabeled control (cells incubatedwith secondary antibody only) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077), at 1/2000 dilution was used as the secondary antibody.