|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.
NOTE: Do not allow slides to dry at any time during this procedure.
1. Deparaffinize/hydrate sections:
1. Incubate sections in three washes of xylene for 5 min each.
2. Wash sections two times in dH2O for 5 min each.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
1. Wash sections in dH2O three times for 5 min each.
|Specificity||PKM2 Rabbit Recombinant mAb detects endogenous level of total PKM2.|
|Background||Pyruvate kinase (PK) is a rate-limiting glycolytic enzyme that catalyzes the irreversible transphosphorylation between phosphoenolpyruvate (PEP) and adenosine diphosphate, which produces pyruvate and ATP. In mammals, the PK family consists of four isoforms: liver-type PK (PKL); red blood cell PK (PKR); and PK muscle isozyme M1 and M2 (PKM1 and PKM2, respectively). These isoforms are encoded by two genes, PK, liver and red blood cell (PKLR) and PK, muscle (PKM). PKM1 and PKM2 are produced by alternative splicing of the primary RNA transcript of the PKM gene. The non-allosteric isoform PKM1 is constitutively active, and expressed in terminally differentiated tissues, including the muscle and brain, which require a large supply of ATP. By contrast, PKM2 is expressed in tissues with anabolic functions, including proliferating cells and cancer cells, and is subject to complex allosteric regulation. Pyruvate kinase muscle isozyme M2 (PKM2) is a limiting glycolytic enzyme that catalyzes the final step in glycolysis, which is key in tumor metabolism and growth. This reaction is a committed step that leads to glycolysis or oxidative phosphorylation of pyruvate.|