FOXO1A 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.
Specimen Preparation (forcultured cell lines, IF-IC)
1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
1. Add theblocking buffer and incubate for 60 min at RT.
FOXO1A Rabbit Recombinant mAb detects endogenous levels of total FOXO1a.
|Background||FOXO1, also known as forkhead in rhabdomyosarcoma, is a transcription factor that plays important roles in regulation of gluconeogenesis and glycogenolysis by insulin signaling, and is also central to the decision for a preadipocyte to commit to adipogenesis. It is primarily regulated through phosphorylation on multiple residues; In its un-phosphorylated state, FOXO1 is localized to the nucleus, where it binds to the insulin response sequence located in the promoter for glucose 6-phosphatase and increases its rate of transcription. However, when FOXO1 is phosphorylated by Akt on Thr-24, Ser-256, and Ser-319, it is excluded from the nucleus, where it is then ubiquitinated and degraded. The phosphorylation of FOXO1 by Akt subsequently decreases the hepatic glucose production through a decrease in transcription of glucose 6-phosphatase.|