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Cell Line Sample Preparation Protocols

Easily prepare extracts from adherent cells with electrophoresis (Laemmli) buffer for SDS/PAGE or with mild detergent for immunoprecipitation.

Cell Line Lysate Preparation Using Laemmli Sample Buffer

The simplest method for preparing cell lysates is to lyse them directly with electrophoresis (Laemmli) sample buffer.

This method releases almost all cellular proteins into the buffer and are readily available for separation by standard SDS/PAGE methods. However, for some downstream applications (notably immunoprecipitation) refer to the protocol, Cell Line Preparation Using Mild Detergents.

This protocol refers to adherent cells but can be easily adapted to cells growing in suspension.

  1. Wash the cell plate with ice-cold PBS. Repeat 3 times.
  2. Place the plate on ice and add cold Sample Buffer (62.5 mM Tris (pH 6.8), 2% SDS, 10% glycerol, and 100 mM DTT) at a ratio of 5 × 106 cells/ml Sample Buffer.
  3. Scrape the dish with a cell scraper and collect the lysate into a microtube.
  4. Boil the sample at 100°C for 5 minutes.
  5. Sonicate the boiled sample for 5 seconds.
  6. Centrifuge the sample at 14,000 rpm for 5 minutes 4°C.
  7. Transfer the supernatant to a clean tube and store at -80°C until further use.
    Note: You can count cells can from a parallel plate by dislodging the cells with trypsin and counting them.

Cell Line Sample Preparation Using Mild Detergents

We recommend cell lysis with a mild detergent such as Triton X-100 or NP-40 for downstream applications like immunoprecipitation.

The protocol refers to lysis of adherent cells but can be easily adapted to cells growing in suspension.

  1. Wash the cell plate with ice-cold PBS. Repeat 3 times.
  2. Place the plate on ice and add ice-cold Lysis Buffer C (50 mM Tris (pH 7.6), 150 mM NaCl, 5 mM EDTA (pH 8), 1% Triton X-100 and protease inhibitor cocktail) at a ratio of 5 × 106 cells/ml Lysis Buffer C.
  3. Scrape the dish with a cell scraper and collect the lysate into a microtube.
  4. Rock the sample in the microtube for 30 minutes at 4°C.
  5. Centrifuge the sample at 14,000 rpm for 10 minutes at 4°C.
  6. Carefully transfer the clear supernatant to a clean microtube. The sample can be stored at -20°C for several months or immediately mixed with50 ml Laemmli sample buffer and loaded onto an SDS/PAGE gel.
    Note: You can count cells from a parallel plate by dislodging the cells with trypsin and counting them.