|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||Cdk6 Rabbit Recombinant mAb detects endogenous level of total Cdk6.|
|Background||A cyclin-dependent kinase (CDK) is a critical regulator of cell-cycle progression, becoming activated upon binding to cyclins. Progression through the G1 phase of the cell cycle is mediated by activation of the CDK4/6-cyclinD complex and subsequent phosphorylation of the retinoblastoma protein, which triggers E2F-dependent transcription. Cdk4 and Cdk6, share 71% amino acid identity and both partner with all three D-type cyclins to phosphorylate pRb. Although the cyclin-dependent kinase 6 (CDK6) and CDK4 have redundant functions in regulating cell-cycle progression, studies of tumors have also demonstrated differences between cdk4 and cdk6. Certain types of tumors selectively amplify either cdk4 or cdk6. Cdk4 and cdk6 also demonstrate distinct patterns of sub-cellular localization. Besides, CDK6 is found to have a new role in the differentiation of a variety of cell types and is apparently not shared with cdk4. CDK6 can block differentiation.|