Dounce homogenizer

Briefly warm at room temperature, and pellet the protein at 10,000g for 10 min at room temperature, pour off the acetone, air-dry the pellet, and redisperse it in saline at 1 mg carrier/mL Since the pelleted protein is rather sticky, this is best done using a Dounce homogenizer. If necessary, store the immunogen at -20°C and rehomogenize before use... 

Acetone precipitates the aqueous immunogen is precipitated with 4.5 vol acetone at -20°C. The precipitate is collected by centrifugation at 10,000 at room temperature, washed in 80% acetone and air-dried. The pellet is resuspended in saline using a Dounce homogenizer and then administered directly or in association with alum or Freund s adjuvants. 

B. For small, soft organisms the tissue can be placed directly into a grinding buffer without liquid nitrogen treatment. The tissue can then be dispersed by grinding in a Teflon Dounce homogenizer. 

Dounce homogenizer with a large clearance pestle 0.06-0.08 mm... 

Transfer the lysate to a 40-ml Dounce homogenizer. Homogenize the lysate with 20 strokes to shear cellular DNA. 

The number of dounce homogenations required depends on the amount of connective tissue within the organ. Dounce until tissue gives little resistance. 

Homogenization Soft animal tissues are homogenized in hypotonic buffer using a Potter-Elvenhjem or Dounce homogenizer by forcing cells through narrow gap between pestle and vessel. 

Unless otherwise noted, all procedures should be performed at 4°C or on ice. Once prepared. Drosophila nuclei should be handled identically, regardless of source. The nucleus-enriched pellet should be resuspended in 20 mM Tris-HCl, pH 7.5,5 mM MgCla (Buffer B). For nuclear resuspension, one volume of Buffer B relative to the initial volume of starting material should be used. For example, if nuclei are purified from 10 ml of embryos, 10 ml of Buffer B should be used if nuclei are purified from 1 ml of tissue culture cells, 1 ml of Buffer B should be used. Complete resuspension should be assured by Dounce homogenization (two-four strokes, loose pestle) and both DNase I and RNase A should be added to final concentrations of 10 and 8 /i.g/ml, respectively. Typically, RNase A is added first, the sample is mixed on a vortex mixer, and then the DNase I is added. After addition of DNase I, vortex mixing is avoided because DNase I is very sensitive to inactivation by oxidation. Rather, the sample is mixed by gentle agitation. 

Homogenize the cells in a Dounce homogenizer with 10 strokes using a tight-fitting pestle (Dounce B). Monitor cell disruption in the phase-contrast microscope. 

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