Product nameHuman BAD knockout HeLa cell lysate
See all Bad kits
Knockout cell lysate achieved by CRISPR/Cas9.
Parental Cell LineHeLa
Mutation descriptionKnockout achieved by using CRISPR/Cas9, Homozygous: Insertion of the selection cassette in exon 2.
Knockout validationSanger Sequencing, Western Blot (WB)
Reconstitution notesTo use as WB control, resuspend the lyophilizate in 50 µL of LDS* Sample Buffer to have a final concentration of 2 mg/ml. For reducing conditions, we recommend a final concentration of 0.1 M DTT.
*Usage of SDS sample buffer is not recommended with these lyophilized lysates.
Lysate preparation: Our lysates are made using RIPA buffer to which we add a protease inhibitor cocktail and phosphatase inhibitor cocktail (ratio: 300:100:10). This means that the protein of interest is denatured. If you require a native form of the protein please use the live cell version - found here. Please refer to our lysis protocol for further details on how our lysates are prepared.
User storage instructions: After reconstitution, store the lysate at -80°C.
Access thousands of knockout cell lysates, generated from commonly used cancer cell lines.
See here for more information on knockout cell lysates.
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It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.
This product is subject to limited use licenses from The Broad Institute, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.
Tested applicationsSuitable for: WBmore details
Storage instructionsStore at -80°C. Please refer to protocols.
Components 1 kit ab260098 - Human BAD knockout HeLa cell lysate (Lyophilized) 1 x 100µg ab255552 - Human wild-type HeLa cell lysate (Lyophilized) 1 x 100µg
STR AnalysisAmelogenin X D5S818: 11, 12 D13S317: 12, 13.3 D7S820: 8, 12 D16S539: 9, 10 vWA: 16, 18 TH01: 7 TPOX: 8, 12 CSF1PO: 9, 10
FunctionPromotes cell death. Successfully competes for the binding to Bcl-X(L), Bcl-2 and Bcl-W, thereby affecting the level of heterodimerization of these proteins with BAX. Can reverse the death repressor activity of Bcl-X(L), but not that of Bcl-2 (By similarity). Appears to act as a link between growth factor receptor signaling and the apoptotic pathways.
Tissue specificityExpressed in a wide variety of tissues.
Sequence similaritiesBelongs to the Bcl-2 family.
DomainIntact BH3 motif is required by BIK, BID, BAK, BAD and BAX for their pro-apoptotic activity and for their interaction with anti-apoptotic members of the Bcl-2 family.
modificationsPhosphorylated on one or more of Ser-75, Ser-99, Ser-118 and Ser-134 in response to survival stimuli, which blocks its pro-apoptotic activity. Phosphorylation on Ser-99 or Ser-75 promotes heterodimerization with 14-3-3 proteins. This interaction then facilitates the phosphorylation at Ser-118, a site within the BH3 motif, leading to the release of Bcl-X(L) and the promotion of cell survival. Ser-99 is the major site of AKT/PKB phosphorylation, Ser-118 the major site of protein kinase A (CAPK) phosphorylation. Ser-75 is phosphorylated by AKT/PKB, protein kinase A and PIM2.
Cellular localizationMitochondrion outer membrane. Cytoplasm. Upon phosphorylation, locates to the cytoplasm.
- Information by UniProt
Our Abpromise guarantee covers the use of ab256847 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use at an assay dependent concentration.|
Lane 1: Wild-type HeLa cell lysate (20µg)
Lane 2: BAD knockout HeLa cell lysate (20µg)
Lanes 1- 2: Merged signal (red and green). Green - ab32445 observed at 23 kDa. Red - loading control ab8245 observed at 37 kDa.
ab32445 Anti-Bad antibody [Y208] was shown to specifically 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 and BAD knockout samples 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) were incubated overnight at 4°C at 1 in 2000 ?g/ml 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.
Homozygous: Insertion of the selection cassette in exon 2
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
ab256847 has not yet been referenced specifically in any publications.