The region of AKT2 surrounding S474 has a high degree of similarity to the corresponding regions in AKT1 and AKT3 and thus may cross react with these proteins if phosphorylated on the corresponding serine residue.
This antibody gave a positive signal in LPS-treated HL60 whole cell lysate.
This antibody gave a positive result in IHC in the following FFPE tissue: Human pancreas adenocarcinoma.
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
pH: 7.40 Preservative: 0.02% Sodium azide Constituent: PBS Note: Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
Use a concentration of 1 µg/ml. Detects a band of approximately 55 kDa (predicted molecular weight: 55 kDa). Abcam recommends using milk as the blocking agent - 3%
Use a concentration of 5 µg/ml.
General protein kinase capable of phosphorylating several known proteins.
Expressed in all human cell types so far analyzed.
Belongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family. RAC subfamily. Contains 1 AGC-kinase C-terminal domain. Contains 1 PH domain. Contains 1 protein kinase domain.
Phosphorylation on Thr-309 and Ser-474 is required for full activity. Ubiquitinated; undergoes both 'Lys-48'- and 'Lys-63'-linked polyubiquitination. TRAF6-induced 'Lys-63'-linked AKT2 ubiquitination. When fully phosphorylated and translocated into the nucleus, undergoes 'Lys-48'-polyubiquitination catalyzed by TTC3, leading to its degradation by the proteasome.
v akt murine thymoma viral oncogene homolog 2 antibody
Western blot - Anti-AKT2 (phospho S474) antibody (ab110231)
All lanes : Anti-AKT2 (phospho S474) antibody (ab110231) at 1 µg/ml
Lane 1 : LPS-Treated HL60 Whole Cell Lysate Lane 2 : LPS-Treated HL60 Whole Cell Lysate with Immunising peptide at 1 µg/ml Lane 3 : LPS-Treated HL60 Whole Cell Lysate with Control peptide at 1 µg/ml
Lysates/proteins at 25 µg per lane.
Secondary All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 55 kDa Observed band size: 55 kDa
Exposure time: 20 minutes
Abcam recommends using milk as the blocking agent (3%). Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.
This blot was produced using a 10% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 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 ab110231 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.
IHC image of AKT2 (phospho S474) staining in Human pancreas adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab110231, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
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.
ab110231 was tested using an Indirect ELISA approach. The wells were coated with peptide (1µg/ml at 100µl/well) overnight at 4°C, followed by a 5% BSA blocking step for 1 hour at room temperature. The primary Ab was then added at a dilution range of 1- 0.00025µg/ml (100µl/well) for 1hr at room temperature. A HRP-conjugated anti-rabbit IgG (heavy and light chain) was used as a secondary antibody at 1:20,000 dilution for 1hr at room temperature.
Hedl M et al. IRF5 and IRF5 Disease-Risk Variants Increase Glycolysis and Human M1 Macrophage Polarization by Regulating Proximal Signaling and Akt2 Activation. Cell Rep16:2442-55 (2016).
Read more (PubMed: 27545875) »