|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.
PRAS40 Rabbit Recombinant mAb detects endogenous levels of total PRAS40.
|Background||Proline-rich Akt substrate 40 kDa (PRAS40) bridges cell signaling between protein kinase B (Akt) and the mammalian target of rapamycin complex 1 (mTORC1). The activity of PRAS40 is closely mediated by phosphorylation. There are several important phosphorylable sites in PRAS40, including serine (Ser) 183, Ser212, and Ser221 in addition to threonine (Thr) 246, involved in the response to different stimuli. Both Akt and mTORC1 can phosphorylate PRAS40. PRAS40 is one of the substrates of Akt, which regulates the activity of PRAS40 through phosphorylation of its Thr246 residue. As a negative regulator of mTORC1, PRAS40 prevents the binding of mTOR to its substrates. The phosphorylation of PRAS40 results in its dissociation from mTORC1 and enhanced mTOR activation. PRAS40 in conjunction with mTORC1 has been closely associated with programmed cell death and is implicated in diabetes mellitus (DM), cardiovascular diseases, cancer, and neurological diseases. Thus, targeting PRAS40 might hold great promise for innovative therapeutic strategies for these diseases.|