|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.
|Specificity||AMPK beta 1 Rabbit Recombinant mAb detects endogenous level of total AMPK beta 1.|
|Background||AMPK is a heterotrimeric protein consisting of one catalytic subunit (α) and two non-catalytic subunits (β and γ). Two isoforms of α and β subunits (α1 and α2, and β1 and β2), and three isoforms of the γ subunit (γ1, γ2 and γ3) have been identified. AMPK is a primary sensor of cellular energy changes. In response to environmental stresses that cause ATP depletion in cells, the kinase activity of AMPK is instantly induced and the increased activity either inhibits ATP consuming pathways or promotes ATP producing pathways. Three mechanisms were identified that contribute to the activation of the kinase: (i) a direct allosteric activation; (ii) activation through phosphorylation by an upstream kinase and (iii) maintaining the activity through inhibition of dephosphorylation. The β1 subunit of AMPK binds to two other subunits, a and g in a 1:1:1 ratio and serves as a ‘bridge' between them to form a stable trimeric complex. β subunit might modulate the kinase activity through regulation of cellular localization of the enzyme. The β1 subunit can be transcripitionally upregulated by cold shock and a DNA damaging agent, etoposide, and such induction is independent of p53. And forced expression of AMPK-β1 inhibits tumor cell growth.|