Anti-Bad antibody [Y208] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bad antibody [Y208] - BSA and Azide free (AB220116)

This data was developed using ab32445, the same antibody clone in a different buffer formulation.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue sections labeling Bad with purified ab32445 at 1/1000 dilution (0.14 μg/mL). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• WB

Unknown

Western blot - Anti-Bad antibody [Y208] - BSA and Azide free (AB220116)

All lanes:

Western blot - Anti-Bad antibody [Y208] (<a href='/en-us/products/primary-antibodies/bad-antibody-y208-ab32445'>ab32445</a>) at 1/2000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 15 µg

Lane 2:

HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate at 15 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: 18 kDa

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• WB

Lab

Western blot - Anti-Bad antibody [Y208] - BSA and Azide free (AB220116)

This data was developed using the same antibody clone in a different buffer formulation (ab32445).

Lanes 1- 2 : Merged signal (red and green). Green - ab32445 observed at 23 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab32445 was shown to react with Bad in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264843 (knockout cell lysate ab256847) was used. Wild-type HeLa and BAD knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab32445 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 2000 dilution and a 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-Bad antibody [Y208] (<a href='/en-us/products/primary-antibodies/bad-antibody-y208-ab32445'>ab32445</a>) at 1/2000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

BAD knockout HeLa cell lysate at 20 µg

Predicted band size: 18 kDa

Observed band size: 23 kDa

false

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Bad antibody [Y208] - BSA and Azide free (AB220116)

This data was developed using ab32445, the same antibody clone in a different buffer formulation.
Immunocytochemistry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Bad with purified ab32445 at 1/50 dilution (2.9 μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. 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)

Lab

Flow Cytometry (Intracellular) - Anti-Bad antibody [Y208] - BSA and Azide free (AB220116)

This data was developed using ab32445, the same antibody clone in a different buffer formulation. Intracellular Flow Cytometry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labelling Bad with purified ab32445 at 1/20 dilution (10 μg/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). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bad antibody [Y208] - BSA and Azide free (AB220116)

This data was developed using ab32445, the same antibody clone in a different buffer formulation.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue sections labeling Bad with purified ab32445 at 1/1000 dilution (0.14 μg/mL). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bad antibody [Y208] - BSA and Azide free (AB220116)

This data was developed using ab32445, the same antibody clone in a different buffer formulation.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue sections labeling Bad with purified ab32445 at 1/1000 dilution (0.14 μg/mL). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bad antibody [Y208] - BSA and Azide free (AB220116)

This data was developed using ab32445, the same antibody clone in a different buffer formulation.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovarian cancer tissue sections labeling Bad with purified ab32445 at 1/1000 dilution (0.14 μg/mL). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• WB

Lab

Western blot - Anti-Bad antibody [Y208] - BSA and Azide free (AB220116)

This data was developed using the same antibody clone in a different buffer formulation (ab32445).

Lanes 1 - 4 : Merged signal (red and green). Green - ab32445 observed at 23 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab32445 was shown to specifically recognise BAD in wild-type HAP1 cells along with additional cross reactive bands. No band was observed when BAD knockout cells were examined. Wild-type and BAD knockout samples were subjected to SDS-PAGE. ab32445 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/2000 dilution and 1/20,000 dilution respectively. Blots were developed 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 1/20,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Bad antibody [Y208] - BSA and Azide free (ab220116) at 1/2000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

BAD knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

MCF7 whole cell lysate at 20 µg

Predicted band size: 18 kDa

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