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Cell Lysates and Tissue Extracts for Malachite Green

Cell lysates and tissue extracts contain millimolar concentrations of free phosphate that will interfere with the phosphatase assay. In addition, ATP at high concentrations can increase the background and lead to phosphorylation by contaminating kinases. It is therefore necessary to eliminate these components from samples by running samples over a Sephadex® G-25 resin (GE Healthcare Biosciences). The method of extraction may influence both the recovery of phosphatases and the presence of inhibitors of these enzymes

Protocol

Sample and Column Preparation

  1. Homogenize the tissue at 0 to 4°C for 30 seconds using 1 g of cells in 3 mL of storage buffer (10 mM Tris-HCL [pH 7.5] 1 mM EDTA 0.02% sodium azide).

    1. Note:The choice of storage buffer depends on several factors, including whether membrane-associated or cytoplasmic phosphatases are being examined. The storage buffer will generally include a reducing agent, a chelator of divalent cations and various protease inhibitors.
    2. Up to 1% detergent (e.g., Triton X-100) can be used to prepare membrane-associated phosphatases if the sample is diluted appropriately in the reaction mix. Here are selected recipes for a variety of phosphatase buffers:
      • Tonks, N.K., Diltz, C.D. and Fischer, E.H. Purification of the major proteintyrosine-phosphatases of human placenta. J Biol Chem 1988; 263:6722.
      • Cohen, P. et al. Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle. Methods Enzymology 1988; 159:390.
      • Zhao, Z. et al. Purification and characterization of a protein tyrosine phosphatase containing SH2 domains. J Biol Chem 1993; 268:2816.
      • Zhao, Z. et al. Purification and characterization of PTP2C, a widely distributed protein tyrosine phosphatase containing two SH2 domains. J Biol Chem 1994; 269:8780
  2. Centrifuge the homogenized lysate at 100,000 xg at 4°C for 1 hour to remove particulate matter.
  3. Add 10 mL of de-ionized water to a spin column and allow it to drain. To begin draining the column the first time, apply slight pressure to the spin column with a 10 mL syringe or shake it briskly. The supplied Adaptor allows centrifugation in most standard 50 mL disposable tubes. (A lid is not necessary.) Allow the spin column to drain into a waste container.
  4. Resuspend the Sephadex G-25 resin by rocking gently or pipetting with a wide-mouth pipette.
  5. Pipette 10 mL of the resuspended Sephadex slurry into the spin column and allow it to drain by gravity into a spare 50 mL tube. Remove the flow-through liquid from the tube.
  6. Add 10 mL of ice-cold phosphatase storage buffer to the column.
  7. Allow the column to drain by gravity, remove the flow-through liquid from the tube, and then centrifuge at 600 xg for 5 minutes at 4°C using a spare 50 mL tube to remove the remaining buffer surrounding the Sephadex beads.
  8. Place the spin column with adaptor in the supplied reservoir (50 mL tube) and add 250 µL of tissue extract or cell lysate. (A larger volume of sample may be used; see Note following Step 9.)
  9. Centrifuge at 600 xg for 5 minutes at 4°C. The sample lysate in storage buffer will be in the bottom of the reservoir in the original volume.

    10. Note: The sample flow-through in the Reservoir should contain 4–10% of the endogenous phosphate. This reduced phosphate level should be low enough to perform most experiments with minimal background. If additional phosphate must be removed, pass the collected sample flow through a second Spin Column.

    For determination of specific activities, measure the protein concentration of the phosphate-reduced sample(s). Using a larger sample volume in Step 8 will reduce the efficiency with which free phosphate is removed. For example, if 500 µL of sample is used in Step 8, approximately 85–90% of the endogenous phosphate will be removed. The protocol above should reduce the high concentration of phosphatase in crude cellular preparations prior to performing the assay. The spin columns rapidly and effectively remove free phosphate and other low molecular weight inhibitors from the sample. In addition, because the assay measures free phosphate, phosphate buffers are not compatible with this system.

    Reaction components that contain phosphate (i.e., glycerol phosphate) may interfere with the analysis, depending on their concentration, purity and stability in strong acid. High concentrations of reductants may bleach the dye color over time resulting in lower sensitivity. A final concentration of 0.02% p-mercaptoethanol has no effect on sensitivity; 0.05% p-mercaptoethanol has only a slight effect, and 0.1% p-mercaptoethanol results in an approximate 20% reduction in sensitivity. Many detergents at or below 0.1% can be used, but higher concentrations may generate high backgrounds. If high concentrations of detergent are required in the reaction, the background can be determined by including the corresponding concentration of the detergent in the Phosphate Standard curve.

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