Sample preparation flash-frozen samples

The tissue of interest should be removed from the animals using equipment that has been treated to ensure that it is RNase-free. The surface hair of the animals can be soaked in 70% ethanol to minimize the inclusion of hair or dander in the isolated tissues. A sterile disposable Petri plate, placed on top of a bed of crushed ice, is a suitable RNase-free surface for microdissection. Immediately after isolation from the animal, individual samples of tissue can be flash frozen by immersion in liquid nitrogen and stored in cryovials in liquid nitrogen or at -70°C until all samples from an experimental set have been obtained. RNA extraction and preparation of single-stranded cDNA can then be performed simultaneously on all samples. This will minimize differences between RNA populations that result from differences in sample preparation. 

Any RNA preparation method can be used that provides intact, undegraded RNA. Biopsy samples can be flash frozen, pulverized, and suspended in guanidinium thiocyanate prior to extraction by the Chomczynski method (24). Small, fresh samples may also be suspended in guanidinium thiocyanate. In order to shear the sample for improved extraction, the samples should be passed several times through a 22-g needle, attached to a plastic syringe. The extraction steps can be repeated several times in order to get a clean RNA preparation prior to precipitation. 

Nowadays, cryo-electron microscopy, so named for the method of sample preparation and maintenance, is by far the most effective technique of electron microscopy for biological macromolecule characterisation. Therefore, we shall focus on this electron microscopy technique alone from this point onwards. The basis of success in cryo-electron microscopy is very simple though practically quite demanding. A sample of biological macromolecule of interest in water is flash frozen in such a way that individual molecules become embedded... 

NE assembly can be assayed in either freshly prepared extracts or frozen extracts. Although we usually use extracts within 30 min of their preparation, extracts can retain the ability to assemble NEs after storage for up to 4 hr on ice. To freeze extracts, 0.2 ml of extract is mixed with 44 /tl of an unbuffered 2.2 M sucrose solution. The sucrose and extract are mixed with a wide-bore pipet tip. After mixing, each sample is immediately flash frozen in liquid nitrogen and stored at —80°C. Both the ratio of extract to sucrose and the total amount of extract frozen in one tube seem to be critical to retain NE assembly activity after the extract has been thawed (M. S. Carpenter, unpublished). Extracts are thawed on ice before use. Not all extracts retain the ability to assemble NEs after they have been frozen and thawed. 

The syringe is withdrawn from the side of the centrifuge tube and the crude NEP-A fraction is transferred to a glass tube on ice. The samples collected from the four tubes are pooled together. The NEP-A fraction is mixed with an equal volume of 60% sucrose, in either GB or PB, depending on which was used to prepare the crude extract. The crude NEP-A/30% sucrose mixture is divided into 0.5-ml samples, flash frozen in liquid nitrogen, and stored at -80°C. 

The cytosol is divided into 0.1-ml samples, flash frozen, and stored at -80°C. A good cytosol preparation will not form nuclear envelopes when mixed with the sucrose-purified NEP-A fraction, nor will it contain vesicles that bind to the sperm chromatin. 

The HX experiment can be automated by following two distinct experimental workflows. In one paradigm, the experimental design decouples automated digestion and LC-MS analysis from the HX sample preparation step (i.e., decoupled HX-MS). To enable the LC-MS analysis to be decoupled from the HX expraiment, samples are flash frozen in liquid nitrogen afta- the quench and stored at -80°C until required. Samples can be stored for days/weeks at -80°C with minimal loss of deuterium label. When the sample is ready for LC-MS analysis, it is thawed immediately prior to analysis with LC-MS by the autosampler. 

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