Anti-CBFb antibody [EPR6322]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CBFb antibody [EPR6322] (AB133600)

Immunocytochemistry/Immunofluorescence analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling CBFb with purified ab133600 at 1/100 dilution (10 μg/mL). Cells were fixed in 100% Methanol and permeabilized with None. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 μg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CBFb antibody [EPR6322] (AB133600)

Intracellular Flow Cytometry analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells, labeling CBFb with purified ab133600 at 1/100 dilution (10μg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^®488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• WB

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Western blot - Anti-CBFb antibody [EPR6322] (AB133600)

Blocking/Diluting buffer : 5% NFDM/TBST

Loading Control : Rabbit monoclonal [EPR16891] to GAPDH (ab181602)

All lanes:

Western blot - Anti-CBFb antibody [EPR6322] (ab133600) at 1/1000 dilution

Lane 1:

Raji (Human Burkitt's lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 4:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Lane 5:

RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 6:

Mouse spleen lysate at 20 µg

Lane 7:

Mouse thymus lysate at 20 µg

Lane 8:

Rat spleen lysate at 20 µg

Lane 9:

Rat thymus 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: 22 kDa

Observed band size: 22 kDa

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• WB

Lab

Western blot - Anti-CBFb antibody [EPR6322] (AB133600)

ab133600 was shown to react with CBFb in wild-type A431 cells in western blot. Loss of signal was observed when CBFB knockout sample was used. Membranes were blocked in 2 % BSA in TBS-T (0.1 % Tween^®) before incubation with ab133600 overnight at 4°C at a 1 in 1000 dilution and ab184095 (Mouse Anti-GAPDH antibody [mAbcam 9484] - Alexa Fluor^® 680) at a 1 in 1000 dilution. Blots were incubated with HRP conjugated Goat anti-Rabbit (H+L) secondary antibody at 1/5000 for 1 hour at room temperature before development with Optiblot ECL reagent (ab133456) and imaging.

All lanes:

Western blot - Anti-CBFb antibody [EPR6322] (ab133600) at 1/1000 dilution

Lane 1:

Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

CBFB knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human CBFB knockout A-431 cell line (<a href='/en-us/products/cell-lines/human-cbfb-knockout-a-431-cell-line-ab270472'>ab270472</a>)

Lane 3:

K562 (Human chronic myelogenous leukemia lymphoblast cell line ) whole cell lysate at 20 µg

Lane 4:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Predicted band size: 22 kDa

Observed band size: 24 kDa

false

Exposure time: 150s

• WB

Lab

Western blot - Anti-CBFb antibody [EPR6322] (AB133600)

Lanes 1 - 4 : Merged signal (red and green). Green - ab133600 observed at 22 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab133600 was shown to react with CBFb in wild-type A431 cells in western blot with loss of signal observed in CBFB knockout sample. Wild-type and CBFB knockout A431 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 2% BSA in TBS-T (0.1% Tween^®) before incubation with ab133600 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CBFb antibody [EPR6322] (ab133600) at 1/1000 dilution

Lane 1:

Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

CBFB knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human CBFB knockout A-431 cell line (<a href='/en-us/products/cell-lines/human-cbfb-knockout-a-431-cell-line-ab270472'>ab270472</a>)

Lane 3:

K562 (Human chronic myelogenous leukemia lymphoblast cell line ) whole cell lysate at 20 µg

Lane 4:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Predicted band size: 22 kDa

Observed band size: 22 kDa

false

• OI-RD Scanning

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OI-RD Scanning - Anti-CBFb antibody [EPR6322] (AB133600)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.