Drosophila buffer preparation

To purify lamins. Drosophila extracts prepared as described above should be supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). Lamins from these extracts should be batch adsorbed to antilamin/protein A/sepharose by incubation overnight at 4°C. The column should then be washed extensively with equilibration buffer and purified lamins recovered from the antilamin affinity column with elution buffer. After elution, the solution containing the lamins should be immediately neutralized with Na2HP04 added to a final concentration of 50 mM. Immunoaffinity-purified lamins should be aliquoted, frozen by immersion in liquid nitrogen, and stored at -70°C until use. 

Fresh embryos can be fractionated in a manner similar to frozen embryos. However, significant numbers of unbroken cells are observed if procedures are followed exactly as outlined above to ensure complete breakage, the nonionic detergent, Triton X-100, should be added to initial buffers (Buffer E or A) at a final concentration of 0.5-1.0% (v/v). Drosophila tissue culture cells are useful for many purposes (e.g., radiolabeling of proteins). Nuclei can be prepared from both Kc and Schneider S2 cells using procedures identical with those described above for fresh embryos (see Smith et ai, 1987 Smith and Fisher, 1989 Mans et al, 1995). Cells should be harvested from exponentially growing cultures. 

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. 

Measure by spectrophotometer the DNA in the 0.12M PB and 0.48Af PB fractions. The single-copy and single-stranded DNA should be in the 0.12M PB fractions. For Drosophila an incubation to C t 200 usually is enough (50% of the input DNA is in the 0,11M PB fractions). Sometimes the spectrophotometer readings are not accurate (the phosphate buffer and/or the HAP may alter readings) you may wish to add some labelled total DNA from the same DNA preparation to follow the reassociation kinetics by scintillation counting. 

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