Anti-CEBP Beta antibody [E299] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CEBP Beta antibody [E299] - BSA and Azide free (AB220813)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling CEBP Beta with Purified ab32358 at 1/600 dilution (1 μg/ml) (red). Cells were fixed with 80% 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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32358).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-CEBP Beta antibody [E299] - BSA and Azide free (AB220813)

Overlay histogram showing HeLa (Human epithelial cell line from cervix adenocarcinoma) cells stained with ab32358 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32358, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabeled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32358).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CEBP Beta antibody [E299] - BSA and Azide free (AB220813)

ab32358, at a 1/50 dilution, staining CEBP Beta in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells by Immunofluorescence.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32358).

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-CEBP Beta antibody [E299] - BSA and Azide free (AB220813)

3T3-L1 (Mouse embryonic fibroblast cell line) cells were plated on cover slips, grown to post confluency and treated with adipogenic cocktail for 16 h. Cells were washed briefly with phosphate buffered saline (PBS) and fixed in methanol at −20°C for 5 min. Cells were then blocked with 1% BSA for 30 min before incubation with ab32358 at a 1/100 dilution at 4°C overnight and incubated with Alexa Fluor^® 488 goat anti-rabbit secondary antibodies (1 : 500) for 1 h at room temperature.

DAPI staining was used for visualizing the nuclei.

Images were acquired with an Olympus FlowView FV1000.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32358).

Image from Sikkeland et al. PLoS One. 2013 Jul 10;8(7):e68249. doi: 10.1371/journal.pone.0068249. Print 2013. Fig S1.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-CEBP Beta antibody [E299] - BSA and Azide free (AB220813)

Immunocytochemistry/ Immunofluorescence analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labeling CEBP Beta with Purified ab32358 at 1 : 500 dilution (1.2 µg/ml). Cells were fixed in 100% Methanol. 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 nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32358).

• IP

Unknown

Immunoprecipitation - Anti-CEBP Beta antibody [E299] - BSA and Azide free (AB220813)

ab32358 (purified) at 1 : 30 dilution (2μg) immunoprecipitating CEBP Beta in NIH/3T3 whole cell lysate.
Lane 1 (input) : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate 10μg
Lane 2 (+) : ab32358 & NIH/3T3 whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab32358 in NIH/3T3 whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1 : 1000 dilution.
Blocking and diluting buffer : 5% NFDM/TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32358).

All lanes:

Immunoprecipitation - Anti-CEBP Beta antibody [E299] - C-terminal (<a href='/en-us/products/primary-antibodies/cebp-beta-antibody-e299-c-terminal-ab32358'>ab32358</a>)

Predicted band size: 36 kDa

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• WB

Lab

Western blot - Anti-CEBP Beta antibody [E299] - BSA and Azide free (AB220813)

This data was developed using the same antibody clone in a different buffer formulation (ab32358).

Lanes 1-3 : Merged signal (red and green). Green - ab32358 observed at 40 and 45 kDa. Red - loading control ab7291 observed at 50 kDa.

ab32358 Anti-CEBP Beta antibody [E299] - C-terminal was shown to specifically react with CEBP Beta in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261771 (knockout cell lysate ab256874) was used. Wild-type and CEBP Beta knockout samples were subjected to SDS-PAGE. ab32358 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) 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-CEBP Beta antibody [E299] - C-terminal (<a href='/en-us/products/primary-antibodies/cebp-beta-antibody-e299-c-terminal-ab32358'>ab32358</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

CEBP Beta knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human CEBPB (CEBP Beta) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-cebpb-cebp-beta-knockout-hela-cell-line-ab261771'>ab261771</a>)

Lane 3:

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 1/20000 dilution

Predicted band size: 36 kDa

Observed band size: 40 kDa,45 kDa

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