The Bcl-2-associated death promoter (BAD) protein is a pro-apoptotic member of the Bcl-2 gene family which is involved in initiating apoptosis. BAD is a member of the BH3-only family, a subfamily of the Bcl-2 family. After activation, it is able to form a heterodimer with anti-apoptotic proteins and prevent them from stopping apoptosis. Anti-Bad - http://www.stjohnslabs.com/bad-antibody-p-91310?filter_name=STJ91797 Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation

1. Result
“A very weak protein band which was assumed to be the Bad protein was detected above the 22KD using anti-Bad
(STJ91797) antibody at 1:250 dilution and a rabbit Alexa fluor 680 secondary antibody). There is another strong band
around 37KD position as it was suggested from the provided data sheet. “ - Jun-Yong Huang, Newcastle University
Gel electrophoresis information：10% polyacrylamide gel, constant voltage 160 V, 60 min.
Transfer information：0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 80Amp.
Lane No. Antigen
Loading
amount
Primary anti-
body
Primary anti-
body dilution
ratio
Secondary
antibody dilution
ratio
Target band KD
Visualiza-
tion time
1 Hela lysate 10 ug STJ91797 1:250 1:1000 ~24KD /
Cell Line: Lysates from Hela cell line
Method of validation: Western Blot
Primary Antibody: STJ91797 anti-Bad antibody
Secondary Antibody
Alexa fluor 680 rabbit IgG
(H+L) antibody
Dilution ratio: 1:250
Protocol
Treatment of materials: When cells were cultured to 70%-80% plating density, lysis buffer with protease inhibitors
(Sigma) and equal volume of 2X loading buffer were added and heating at 100˚C for 10 min. Supernatant was collected
after centrifuged at 12,000 rpm for 25 min at 4oC and used for 10% Bis-Tris plus gel for protein separation.
E.g. Gel electrophoresis: 0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant current 80 Amp 90 min.
Transfer: 0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 min.
Blocking: 1X Odyssey blocking solution for 2 hr at RT.
Primary antibody probing: primary antibody was added to the 1X Odyssey blocking solution with 1:250 dilution, and
the membrane was incubated at 4˚C for overnight with agitation
Membrane wash: 1X TBST wash for 3 times.
Secondary antibody probing: secondary antibody was added to 1X Odyssey blocking solution with 1:500 dilution with
1:1000 dilution ratio, for 1.5 hr at RT
Membrane wash: 1X TBST wash for 3 times.
Visualization: : Fluorescent signals were visualization using a Li-Cor Odyseey machine using 700nm and 800nm de-
tecting channels.
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STJ91797 Anti-Bad Antibody
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