Proteinase-K digestion

Variations Other protocols use high-salt solutions in combination with isopropanol to precipitate RNA from sucrose fractions. Standard proteinase K digestion in the presence of SDS may be used to extract RNA from the precipitated material. 

PrPres Prion protein that is partially resistant against proteinase K digest (in most cases equal to PrPSc or PrP27-30)... 

Although strongly suggestive, these experiments do not prove that the normal pathway of infection involves only the endocytic route. Infection could also occur by fusion of the virus with the plasma membrane. This was shown not to be the case by allowing uptake of SFV into BHK-21 cells for 10 minutes at 37°C in the presence of inhibitory concentrations of NH4CI (Helenius et al., 1982). All of the viruses left on the cell surface were then removed by proteinase K digestion at 0°C, and after removal of the inhibitor, the incubation continued at 37°C. The intracellular viruses were shown to infect the cells almost as efficiently as in control cells. 

Refix the tissue in freshly prepared 4% paraformaldehyde for -4 h after xylene treatment and before proteinase K digestion (see Note 13). 

Optimize conditions for proteinase K digestion by adding 250 J,L of different concentrations of the enzyme on different slides of the same tissue block. Incubate for 15 min at 37°C. 

Select the following tissue slides (sectioned from one tissue block) after proteinase K digestion one with suboptimal digestion one with optimum digestion and one with over digestion. 

Select slides with optimized proteinase K digestion and follow the steps below for DNase digestion. Prepare in an Eppendorf tube 1.0 pL RNasin (RNase inhibitor), 11.5 pL UV-irradiated dH20, 37.5 U DNase (RQl RNase free DNase, Promega Inc., Madison, Wl)/(1 U/pL) Total (per slide) 50.0 pL. 

It is important to proceed further only if tissue retains its morphology intact after proteinase K digestion. Discontinue the experiment if the tissue is distorted. This saves your valuable time and reagents. 

In intermediate rodent hosts, the PCR is routinely used in epidemiological studies to identify E. multilocularis from liver lesions, which are often very small, atypical or calcified. The PCR using proteinase K-digested lesion material has been successfully employed for monitoring the prevalence of E. multilocularis in rodents (Dinkel et al., 1998 Gottstein et al., 2001 Stieger et al., 2002). 

Abubakar, A., Saito, T., Kitazawa, H., Kawai, Y., and Itoh, T. 1998. Structural analysis of new antihypertensive peptides derived from cheese whey protein by proteinase K digestion. J. Dairy Sci. 81, 3131-3138. 

Fast and simple methods for extraction of DNA or RNA from cells, whole blood, and other fluids have been reviewed by Bloch24 and Kawasaki.25 In general, target DNA is released from the cells or virus by proteinase K digestion in the presence of detergent followed by heat inactivation of the proteinase K. A small aliquot of the cell lysate is then used directly as the template in the amplification reaction. For cell culture supernatant fractions, the proviral DNA released from the lysed cells can be directly... 

A first step in the LC-MS characterization is the solubilization of the membrane proteins, which may be achieved by chemical and/or (multi-)enzymatic digestion in combination with the nse of snrfactants, organic solvents, and/or acids. High pH treatment and nonspecific proteinase K digestion has also been proposed [77]. 

Purified GPIs, obtained by proteinase K digestion from the mucins of T. cruzi trypomastigotes (tGPIs), are potent inducers of nitric oxide, tumor necrosis factor-a and interleukin-12. The unsaturated fatty acid of the GPI alkylacyl-PI moiety is essential for bioactivity, but other features of the intact GPI are also necessary since the PI obtained by nitrous acid treatment was inactive.32 Correlation between GPI structure and proinflammatory activity in macrophages has been reviewed.52... 

The DNA extraction method is an in-house developed protocol. Microdissected tumor area are dipped into xylene to remove paraffin, rehydrated in a series of ethanol, and incubated in a proteinase K digestion buffer. Two different DNA purification methods are used. The first method is a NaCl saturated solution precipitation, as previously described. In the second method we use reagents and materials provided by the QIAamp DNA Blood... 

The combination of proteinase K digestion of bacterial proteins followed by nuclease digestion and phenol water extraction results in an LPS preparation of very high quality, free of contaminating proteins and nucleic acids. 

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