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Sample Preparation for Microinjection

  • Microinject all cells on the coverslip.
  • Proceed with biochemical analysis (depends on the particular experiment).
  • Note: For time series experiments, the cells may be plated directly on to Eppendorf CELLocate gridded cover slips placed in the center of a glass slide. Thereby individual cells (55 m) or cell groups (175 m) can be located easily after microinjection. Eppendorf Micromanipulator 5179 and FemtoJet can be used with Femtotips and Femtotips II precision microcapillaries to efficiently perform cytoplasmic and nuclear microinjection on a wide range of adherent cells. Proteins, Antibodies Purification[4]Purification methods which result in the highest activity of the particular proteins or antibodies should be used. For peptide antibodies raised in rabbits, affinity purification is recommended. The concentration of the material injected should be 10 to 20 times higher than that required for the optimum in vitro activity, because the sample is diluted 10 to 20 times when injected into the cells.
    Storage Purified antibodies must be stored in the concen-tration in which they arise. Shock freeze small aliquots of 510 l in liquid nitrogen. Store at maximum temp. of 20C (80C is even better). Azide should not be used. Refrain from repeated freezing and defrosting as antibodies lose activity and st
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