A method for isolating respiring mitochondria from epiphyseal cartilage

Enzymic and ultrasonic methods for isolating a respiring mitochondrial fraction from chick epiphyseal cartilage were evaluated. It was found that sonication was the method of choice. Utilizing the “Polytron,” a fraction with elevated cytochrome oxidase activity was obtained. The effects of ADP, DNP, and oligomycin on oxygen consumption indicated that mitochondria in this fraction were biochemically intact and were performing coupled oxidative phosphorylation.     

A simple procedure for isolating adenosine triphosphatase from mitochondria.

A simple method for isolation of adenosine triphosphatase (EC 3.6.1.3) from mitochondria is described. The enzyme is released from mitochondrial Lubrol particles by drastic sonication and purified by gel filtration on Sepharose 6-B. The described procedure is effective in isolating adenosine triphosphatase from rat liver as it is from beef heart mitochondria. The enzyme isolated from beef heart has a specific activity of 120 mumol P/min per mg protein and enzyme isolated from rat liver has a specific activity of 70 mumol P/min per mg protein when measured as a release of inorganic phosphate.     

An improved method for isolating RNA from porcine adipose tissue.

In the present study, we describe a method that we developed to isolate total RNA from porcine adipose tissue. This method entails homogenizing porcine adipose tissue in 10 ml of 4 M guanidium thiocyanate, 25 mM sodium citrate, 0.5% Sarcosyl, 0.1 M beta-mercaptoethanol, pH 7.0, and then performing two CHCl3 extractions to remove lipid before following the procedure described by P. Chomczynski and N. Sacchi (1987, Anal. Biochem. 162, 156-159). This modification improved the yield of RNA approximately threefold (yield was 88 +/- 7 micrograms total RNA/g of tissue) without affecting RNA quality.     

A simple procedure for isolating adenosine triphosphatase from mitochondria

A simple method for isolation of adenosine triphosphatase (EC 3.6.1.3) from mitochondria is described. The enzyme is released from mitochondrial Lubrol particles by drastic sonication and purified by gel filtration on Sepharose 6-B. The described procedure is effective in isolating adenosine triphosphatase from rat liver as it is from beef heart mitochondria. The enzyme isolated from beef heart has a specific activity of 120 μmol P/min per mg protein and enzyme isolated from rat liver has a specific activity of 70 μmol P/min per mg protein when measured as a release of inorganic phosphate.     

A rapid, efficient method for isolating DNA from yeast

A method is described for the purification of chromosomal and plasmid DNA from the yeast Saccharomyces cerevisiae. This method is rapid, gives 75% of theoretical yield, and produces DNA that can be cut with restriction endonucleases. Yeast cells are treated with zymolyase, and the resulting spheroplasts are lysed in the presence of the chaotropic agent guanidine hydrochloride. After a brief ethanol precipitation, protein is removed by treatment with proteinase K followed by phenol-chloroform extraction. After ethanol precipitation, the DNA is sufficiently pure for restriction analysis or for the transformation of Escherichia coli.     

A rapid,simple method for isolating pinocytotic vesicles and plasma membrane of lung

We have found means of isolating pinocytotic vesicles and attached plasma membrane from the low speed (200 × g) supernatant of homogenates of lung. In lung, 5′-nucleotidase is restricted to pinocytotic vesicles and areas of incipient vesicle formation along the plasma membrane. In our method, P_i released from AMP is precipitated as lead phosphate at the subcellular site of 5′-nucleotidase. The resulting increase in density allows collection of pinocytotic vesicles and attached plasma membrane as a pellet after centrifugation through sucrose (d = 1.18) at 250 × g. The final pellet contains long strands of plasma membrane, and the vesicles retain their characteristic morphology including the delicate diaphragm covering their stomata. The entire procedure can be performed in less than 90 min.     

An improved method for isolating Schwann cells from postnatal rat sciatic nerves

The major difficulty in Schwann cell (SC) purification is contamination by fibroblasts, which usually become the predominant cell type during SC enrichment in vitro. Current reported measures are mainly limited by either high cost or complicated procedures with low cell yields or purity. Our objectives have been to develop an efficient, easily applicable, rapid method to obtain highly purified SC from the sciatic nerve of newborn rats. The method involves two rounds of purification to eliminate fibroblasts with the novel combined use of cytosine-B-arabinoside hydrochloride (Ara-C) action and differential cell detachment. Cultured cells were first treated with Ara-C for 24 h. The medium was replaced with the growth medium containing 20 ng/ml human heregulin1-β1 extracellular domain (HRG1-β1 ECD). After another 48 h in culture, the cells were treated with 0.05% trypsin, following which SCs, but not fibroblasts, were easily detached from the dishes. The advantage of this method is that the two steps can eliminate the fibroblasts complementarily. Ara-C eliminates most of the fibroblasts growing among SCs, whereas the differential cell detachment technique removes the remainder growing under or interacting with the SC layer. A purity of more than 99% SCs has been obtained, as confirmed by cell morphology and immunostaining. The purified SCs have a spindle-shaped, bipolar, and sometimes tripolar morphology, align in fascicles, and express S-100. The whole procedure takes about 10 days from primary culture to the purified SCs growing to confluence (only half the time reported previously). This protocol provides an alternative method for investigating peripheral nerve regeneration and potentially could be used to produce enough SCs to construct artificial nerve scaffolds in vitro. This work was supported by Tsinghua-Yue-Yuen Medical Sciences Fund, the National Natural Science Foundation of China (contract grant numbers: 30670528, 30700848, 30772443), Beijing Municipal Science & Technology Commission (BMSTC, contract grant number: H060920050430), National Basic Research Program of China (also called the 973 Program, contract grant number: 2005CB623905), and the National Natural Science Foundation of Beijing (contract grant number: 7082090).     

A method for isolating fungi from soil microhabitats

Summary A technique is described for the separation and washing of soil micro-habitats (the separation being on a size basis). Tests of the efficiency of the washing technique show that, for the soil under experiment, 25 to 30 washings are required to remove most of the fungal spores; the number of washings required will vary from soil to soil. Data is given on the use of this method for fungal isolations from a cultivated soil.     

A method for isolating nuclei from Euglena gracilis

Summary A method is given for isolating nuclei from Euglena gracilis. The method yields 29% of the total DNA in the original culture in the final nuclear pellet. The protein: DNA mass ratio of the final pellet is 2.38.     

A simple method for isolating osteoblasts from mouse calvaria

A method for isolating osteoblasts from mouse calvaria, based on the capability of these cells to migrate onto plastic substrates, is reported. The osteoblasts migrate from small (1-2 mm2) regularly cut fragments of calvaria directly onto plastic surface as a continuous cellular sheet. Isolated osteoblasts retain a poligonal shape and the ability to initiate bone matrix formation in presence of B-glycero phosphate. The method is simple and provides a large quantity of osteoblasts ready to be used directly without the need of being detached and reseeded.     

A method for isolating total RNA from pear leaves

Isolation of high quality RNA fromRosaceae species is particularly difficult. These plants contain considerable amounts of plant polyphenolic compounds and polysaccharides that copurify with RNA, often rendering it unsuitable for either cDNA synthesis and/or hybridization in northern analyses. We describe a method for RNA isolation from pear leaves that is modified from that of Manning (1990). The procedure includes i) an extraction with phenol and PVPP, to remove proteins and polyphenols ii) two purifications by LiCl, with a 2-butoxyethanol treatment between the LiCl steps. The method results in high quality RNA suitable for RT-PCR and northern blot experiments.     

A simple and effective method for isolating RNA from alfalfa pollen

Extraction of RNA from alfalfa pollen using conventional grinding devices resulted in low yields of degraded RNA; degradation appeared to be related to the length of time required to break open the pollen grains with such devices. A glass syringe was modified to provide the restricted, shearing environment necessary for rapidly rupturing this type of tissue. This apparatus would be useful for extracting a variety of molecules from pollen of any species, but is particularly valuable for species which produce low amounts of relatively small pollen grains.