Phospho-Akt(Ser473) Rabbit Recombinant mAb

Catalog No.A5030

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  1. 1. Why choose rabbit monoclonal antibody?
  2. Rabbit monoclonal antibody has over 100 times higher affinity than that of mouse monoclonal.
  3. 2. Why choose recombinant antibodies?
  4. Recombinant antibodies are known for higher purity and minimal deviation between batches, versus regular antibodies.
  • WB

Usage Information

Application WB
Dilution
WB
1:1000
Reactivity Human Mouse Rat
MW (kDa) 60kDa
Source Rabbit
Concentration 1mg/ml
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.

Protocol

WB
+ Expand

Western Blotting

Sample preparation

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

a. Blocking

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.

Description

Specificity

Phospho-Akt(Ser473) Rabbit Recombinant mAb detects endogenous levels of phospho-Akt(Ser473).

Background

AKT, a serine/threonine protein kinase, also known as protein kinase B and a member of AGC family, regulates a variety of cellular processes including survival, proliferation, protein translation and metabolism. AKT contains a pleckstrin homology (PH) domain which binds to PIP3 (phosphatidylinositol (3,4,5)-trisphosphate, PtdIns(3,4,5)P3) in the plasma membrane with high affinity. Once in correct position in the membrane, AKT can be phosphorylated by 3-phosphoinositide dependent protein kinase 1 (PDK1) at threonine 308 (Thr308) residue. Phosphorylation of serine 473 (Ser473) residue is a target of the mTOR complex 2 (mTORC2) and DNA-dependent protein kinase (DNA-PK). Maximal AKT activity is dependent on the phosphorylation status of both Thr308 and Ser473 residues.

Datasheet & MSDS

Related Antibodies

Akt Signaling Pathway Map

Related Akt Products

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