Quantification of Protein and RNA Levels
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Variable analysis
- Trypsinization for cell harvesting
- RIPA lysis buffer for total protein extraction
- Protein levels verified by Western blot
- RNA levels verified by Northern blot
- Cultured cells
- Loading buffer composition (50 mM Tris·HCl, pH 6.8, 2% SDS, 6% Glycerol, 2 mM DTT, 0.01% Bromophenol Blue)
- Denaturation of protein samples (heated at 95 °C for 5 min)
- Protein separation by SDS-PAGE
- Transfer of separated proteins to PVDF membrane
- Staining with specific primary and HRP-conjugated secondary antibodies
- RNA extraction with TRIzol™ Reagent
- Separation of cytosolic fractions of RNAs by denaturing agarose electrophoresis
- Transfer of RNAs to nylon blotting membranes
- Probing with digoxigenin (DIG) labelled Fluc- or Rluc-specific probes
- Anti-beta-actin antibody
- No information provided
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