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

All lanes : Anti-beta 2 Microglobulin antibody [EPR21752-214] (ab218230) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : B2M knockout HEK-293T cell lysate

Performed under reducing conditions.

Predicted band size: 14 kDa
Observed band size: 12 kDa why is the actual band size different from the predicted?

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

Lane 1 : Wild-type HepG2 cell lysate
Lane 2 : B2M knockout HepG2 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : Jurkat cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 14 kDa
Observed band size: 14 kDa

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).

Heat mediated antigen retrieval was performed using 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 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. 

 

 

All lanes : Anti-beta 2 Microglobulin antibody [EPR21752-214] (ab218230) at 1/1000 dilution

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate
Lane 2 : Jurkat (human T cell leukemia T lymphocyte), whole cell lysate
Lane 3 : HepG2 (human liver hepatocellular carcinoma cell line) cell lysate
Lane 4 : Mouse serum
Lane 5 : Mouse plasma
Lane 6 : Rat serum
Lane 7 : Rat plasma
Lane 8 : Human plasma
Lane 9 : Human skin lysate
Lane 10 : Human kidney lysate
Lane 11 : Human liver lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lanes 1-3 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Lanes 4-11 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 14 kDa

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.

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.

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.

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.