Anti-Bim antibody [Y36] - BSA and Azide free (ab170589) + A431 (human epidermoid carcinoma) whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

Predicted band size: 22 kDa


Exposure time: 3 minutes

Blocking buffer and concentration: 5% NFDM/TBST

Diluting buffer and concentration: 5% NFDM/TBST

Clone Y36 (ab170589) has been successfully conjugated by Abcam. This image was generated using Anti-Bim antibody [Y36] (Alexa Fluor® 488). Please refer to ab200667 for protocol details.

ab200667 staining Bim in MCF7 cells. The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab200667 at 1/200 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 2µg/ml (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Overlay histogram showing HAP1 wildtype (green line) and HAP1-BCL2L11 knockout cells (red line) stained with ab32158. The cells were fixed 80% methanol (5 min), and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab32158, 1µg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) presorbed (ab150081) at 1/2000 dilution for 30 min at 22°C.

A rabbit IgG1 isotype control antibody  (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-BCL2L11  knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32158).

ab32158 staining Bim in human breast cancer tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Antigen retrieval was by heat mediated antigen retrieval using Tris/EDTA Buffer, PH9 (ab93684). Samples were incubated with primary antibody (1/100 in blocking buffer) and a Biotin-conjugated Donkey anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody. Cytoplasmic staining can be seen in the human breast cancer cells. Hematoxylin was used as a counter stain.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32158).

Ab32158 at 1/50 immunoprecipitating Bim in Raji (human Burkitt's lymphoma) whole cell lysate.

Lane 1 (input): Raji whole cell lysate (10µg)

Lane 2 (+): ab32158 + Raji whole cell lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32158 in Raji whole cell lysate.

For western blotting, ab32158 (1/1000) was used as the primary antibody and VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

 

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32158).

Flow Cytometry analysis of Raji (human Burkitt's lymphoma) whole cell lysate labeling Bim with ab32158 at 1/100 (red). Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 488)-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG (ab172730). Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32158).

Immunocytochemistry/Immunofluorescence analysis of Raji (Human Burkitt's lymphoma cell line) labeling Bim with ab32158 at a dilution of 1/250. Cells were fixed with 100% methanol. Ab150077 (1/1000) was used as the secondary antibody. Cells were co-stained with ab7291, a mouse anti-tubulin antibody (1/200) using ab150120 as the secondary. Nuclei were counterstained with DAPI (blue).

Secondary antibody only control, cells without incubation with the primary antibody was used as negative control.

Confocal image showing cytoplasmic staining on Raji cell line

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32158).

Immunocytochemistry/Immunofluorescence analysis of A20 (Mouse reticulum sarcoma cell line) labeling Bim with ab32158 at a dilution of 1/250. Cells were fixed with 100% methanol. Ab150077 (1/1000) was used as the secondary antibody. Cells were co-stained with ab7291, a mouse anti-tubulin antibody (1/200) using ab150120 as the secondary. Nuclei were counterstained with DAPI (blue). 

Secondary antibody only control, cells without incubation with the primary antibody was used as negative control.

Confocal image showing cytoplasmic staining on A20 cell line

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32158).

Flow Cytometry analysis of A431 (human epidermoid carcinoma) cells labelling Bim with ab32158 at 1/50 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32158).