HRP Anti-Bak antibody [Y164]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - HRP Anti-Bak antibody [Y164] (AB199516)

IHC image of Bak staining in a section of formalin-fixed paraffin-embedded normal human colon*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins, and incubated overnight at +4°C with ab199516 at 1/125 dilution. DAB was used as the chromogen (ab103723), diluted 1/100 and incubated for 10min at room temperature. The section was counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• WB

Lab

Western blot - HRP Anti-Bak antibody [Y164] (AB199516)

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab199516 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.

All lanes:

Western blot - HRP Anti-Bak antibody [Y164] (ab199516) at 1/5000 dilution

All lanes:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Predicted band size: 23 kDa

Observed band size: 25 kDa

true

Exposure time: 20min

• WB

Lab

Western blot - HRP Anti-Bak antibody [Y164] (AB199516)

ab199516 was shown to specifically react with Bak in wild-type HAP1 cells as signal was lost in BAK1 knockout cells. Wild-type and BAK1 knockout samples were subjected to SDS-PAGE. ab199516 was incubated overnight at 4°C at 1/5000 dilution. Blots were developed with ECL technique.

All lanes:

Western blot - HRP Anti-Bak antibody [Y164] (ab199516) at 1/5000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

BAK1 knockout HAP1 whole cell lysate at 20 µg

Predicted band size: 23 kDa

Observed band size: 25 kDa

true

Exposure time: 20min