Mouse Anti-Akt1, PH Domain, Serine-Threonine Phosphorylation (Rac PKa, PKBa) (MaxLight 650)
MaxLight™650 is a new Far-IR stable dye conjugate comparable to Alexa Fluor™647, DyLight™649, Cy5™ and offers better labeling efficiency, brighter imaging and increased immunodetection. Absorbance (655nm); Emission (676nm); Extinction Coefficient 250,000.
The serine/threonine kinase Akt1 has also been described as Rac-PKa or PKBa. Rac protein kinase family members feature a pleckstrin homology (PH) domain at the amino terminus and a protein-serine/threonine kinase catalytic domain at the carboxy terminus. The PH domain (amino acids 1–106) has been implicated in interactions with other proteins such as G-protein beta gamma subunits, as well as phosphoinositides. The kinase domain is located between residues 148 to 411. These enzymes are activated by diverse ligands such as PGDF, EGF and basic FG in NIH 3T3, Rat-1 or Swiss-3T3 cells. Akt1 is a protein of 480 amino acids and has been observed to show different migratory patterns on a western blot according to the state of phosphorylation of the protein. Phosphatase treatment has been shown to result in inactivation of the protein.
Applications
Suitable for use in Flow Cytometry, Western Blot, Immunocytochemistry, Immunoprecipitation Kinase and Immunoprecipitation. Other applications not tested.
Recommended Dilution
Western Blot Analysis: 0.1-1ug/ml detects Akt (60kD) in RIPA lysates from A431 cells. A431 cell lysate was resolved by electrophoresis, transferred to nitrocellulose and probed with A1125-04 (0.5 and 1.0ug/ml). Proteins were visualized using a goat anti-mouse secondary antibody conjugated to HRP and a chemiluminescence detection system. Immunoprecipitation: 4ug immunoprecipitates Akt from 500ug of L6 RIPA lysate. Immunoprecipitation Kinase: 4ug immunoprecipitates active Akt from 700ug of L6 cells stimulated with 15nM IGF-1 for 2-5 min. Optimal dilutions to be determined by the researcher.
Positive Antigen Control Included
A0001-50: A431 Cells, Non-Stimulated in RIPA Buffer, Sample Buffer. Add 2.5ul of 2-mercaptoethanol/100ul of lysate and boil for 5 minutes to reduce the preparation. Load 20ug of reduced lysate per lane for minigels.
Recommended Secondary Antibodies
I1904-06C: IgG, H&L (HRP) (X-Adsorbed) Pab Gt xMo I1904-06H: IgG, H&L (HRP) (X-Adsorbed) Pab Gt xMo I1904-14C: IgG, Fc (HRP) Pab Gt xMo I1904-19C: IgG, F(ab’)2 (HRP) Pab Gt xMo
Storage and Stability
Store product at 4°C in the dark. DO NOT FREEZE! Stable at 4°C for 12 months after receipt as an undiluted liquid. Dilute required amount only prior to immediate use. Further dilutions can be made in assay buffer. Caution: MaxLight™650 conjugates are sensitive to light. For maximum recovery of product, centrifuge the original vial prior to removing the cap.
Note: Applications are based on unconjugated antibody.
Immunogen
GST-fusion protein corresponding to residues 1-149 of human Akt1
Form
Supplied as a liquid in PBS, pH 7.2. No preservative added. Labeled with MaxLight™650.
Purity
Purified by Protein G affinity chromatography.
Specificity
Recognizes human Akt1/PKBa > Akt2/PKBb (30%) > Akt3/PKBg (weak to none) based on Western blot analysis using 100ng recombinant fusion protein for each isoform. |Species Crossreactivity: mouse and rat.
