PI3 Kinase p85 alpha Rabbit Recombinant mAb
- 1. Why choose rabbit monoclonal antibody?
- Rabbit monoclonal antibody has over 100 times higher affinity than that of mouse monoclonal.
- 2. Why choose recombinant antibodies?
- Recombinant antibodies are known for higher purity and minimal deviation between batches, versus regular antibodies.
|Reactivity||Human Mouse Rat|
|Storage buffer||10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.|
|Storage||Store at –20°C.|
1. Aspirate media from cultures and Wash the cells with 1X PBS. 2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice. 3. Sonicate for 10–15 sec to complete cell lysis and shear DNA. 4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice. 5. Centrifuge for 5 min (with Microcentrifuge). 6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins). NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer. 7. Electrotransfer to nitrocellulose/PVDF membrane.
Membrane Blocking and Antibody Incubations
1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature. 2. Incubate the membrane in the blocking buffer for 1 hr at room temperature. 3. Wash three times for 5 min each with TBST.
b. Antibodies Incubation
1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C. 2. Wash three times for 5 min each with TBST. 3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature. 4. Wash three times for 5 min each with TBST. 5. Proceed with detection.
Detection of Proteins
1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST. 2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well. 3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.
PI 3 Kinase p85 alpha Antibody detects endogenous levels of total PI 3 Kinase p85 alpha.
The phosphoinositide 3-kinase (PI3K) signaling pathway critically regulates cell growth and survival. Class IA phosphoinositide 3-kinase (PI3K) is a heterodimeric enzyme composed of a catalytic subunit (p110) and a regulatory subunit (p85). The regulatory p85 subunit consists of several domains including the SH3 domain, two proline rich fragments, and two SH2 domains separated by the inter-SH2 (iSH2) sequence. The PI3K-Akt pathway is activated by binding of PI3K (p85α) to tyrosine-phosphorylated sites on growth factor receptors or on associated adaptor proteins such as IRS-1 (insulin receptor substrate 1). p85α has diverse functions. As a positive regulator, upon receptor activation, p85α participates in trafficking of p110 to the cell membrane as the initial step in the PI3K-AKT signaling cascade. As a negative regulator, p85α mediates basal inhibition of the p110 subunit. Furthermore, monomeric p85α competes with p85-p110 dimers for activated receptor binding. PI3K plays a critical role in insulin signalling and the PI3K (p85α) exerts both positive and negative effects on the insulin transduction pathways. In some cell types p85 is in excess of p110, and free, monomeric PI3K (p85α) acts as a negative regulator of PI3K signalling. In its unbound form, PI3K (p85α) sequesters the adaptor protein IRS-1 forming cytosolic foci free of p110 and therefore limits the extent of PI3K signaling downstream of the insulin and IGF-1 receptors. The SH3 domain located in the N-terminal portion of PI3K (p85α) is responsible for the interaction with IRS-1.