Proteinase K method

A common method for RNA isolation is the proteinase K method. In this method, the cells are lysed by incubation in a hypotonic solution followed by centrifugation to remove DNA and cell debris. Treatment with the proteolytic enzyme proteinase K leads to the dissociation of RNA-protein complexes and the digestion of the proteins. The digestion products are then removed by phenol/chloroform extraction and the RNA in the remaining aqueous solution is precipitated using ethanol. 

Two procedures can suffice for the isolation of LPS preparations of high purity. These are the modified phenol-water method (13) and a modification of the proteinase K method and phenol extraction, which allows extraction of a LPS preparation free of contaminating lipoproteins (14). 

Rupp and Locker3 Rat liver Homogenized in a solution containing SDS, and proteinase K Not available, using hybridization including dot-blot method RNA purified from FFPE tissue is suitable for hybridization. 

In this method the cells are lysed by incubation in a hypotonic solution, which leaves the nuclei intact. The cell debris and nuclei are pelleted by centrifugation leaving the cytoplasmic RNA free from DNA in the supernatant. The RNA is released from the polysomes by incubation with proteinase K and the protein extracted into phenol/chloroform. The RNA is then precipitated from the aqueous phase using ethanol. 

We have noted that the proteolysis step appears to be the most critical m the whole technique Variability m signal intensity is virtually always caused by incomplete or excessive proteolysis, the only remedy is repetition of the experiment Silane is an effective adhesive for most cells and sections, although occasionally, high concentrations of proteinase K can cause repeated section dehiscence. Under these circumstances, reduction of the proteinase K concentration or, alternatively, the use of pepsin HC1 may prove more effective these, however, reduce the sensitivity of the method. 

Due to phase variation, there are fluctuations in expression levels of certain enzymes in bacteria, therefore, not all colonies or cells make the same structure of lipid A species. A micro-extraction method for extraction of lipid A from a single colony has been developed (Zhou et al., 2009). This method uses microwave-assisted enzymatic digestion and sodium acetate hydrolysis, suitable to analyze lipid A from both cell samples and an individual colony. Because the clean up of SDS is very time-consuming, and the contaminated SDS would seriously interfere with the analysis by mass spectrometry, the proteinase K, instead of SDS, is used to disrupt the cells. Using this method, the entire process for lipid A preparation only takes about 2 h with a detection limit to 1 (xg. 

Some antigens are more efficiently retrieved by a combination of heating and enzyme digestion, e.g. some cytokeratins and immunoglobulin light chains. The protocol below describes a method of first treating with Proteinase K and then HIER by either water bath or microwave method. 

The authors of the studies cited above found that the enzymes used during sample preparation could not completely hydrolyze sample proteins into amino acids. This is partly understandable as both proteinase K and subtilisin are known to show hydrolyzing preference for specific residues of proteins. Accordingly, they do not necessarily arrive at total hydrolysis. At the same time, neither pronase E nor protease XIV could always provide 100 percent extraction of Se from the samples (see Table 19.1). Therefore, alternative methods should be developed in the field of sample preparation to replace enzymatic methods. 

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... 

Fig. 4.2. Isolation of environmental DNA from soils and sediments by the direct lysis approach. The DNA isolation of soil and sediment samples is based on the direct lysis method of Zhou et al. [16]. 50 g of each environmental sample are mixed with 135 ml of DNA extraction buffer (100 mM Tris/HCI, pH 8.0,100 mM sodium EDTA, 100 mM sodium phosphate, 1.5 M NaCI, 1 % (w/v) CTAB) and 1 ml of proteinase K (10 mg/ml) in GS3 tubes by horizontal shaking for 30 min at 37°C. Subsequently 15 ml of 20 % (w/v), SDS is added and the samples are incubated in a 65 °C water bath for 2 h with gentle end-over-end inversion every 15 to 20 min. After centrifugation at 6000 x g for 10 min at room temperature the resulting supernatants are transferred into new GS3 tubes. The remaining pellets are extracted two more times by suspending them in 45 ml of extraction...

Tris-EDTA (TE) buffer 10 mMTris-HQ, 1 mMEDTA, pH 8. Paraformaldehyde (4%, w/v) Boil 100 mL of PBS containing 4 g of paraformaldehyde in a fume cupboard. Cool on ice prior to use. The final pH of this solution should be 7.2-7.4 without adjustment. Proteinase K/pepsin The source used is important The activity of these enzymes varies according to manufacturer and lot We have found the proteinase K supplied by Boehringer Mannheim (FRG) the most consistent, and the details quoted in Methods refer to that source. We find that pepsin obtained from Sigma (Poole, Dorset UK) is superior to that from Boehringer (FUG) and can be used at 0.1% (w/v) compared with 0.4% (w/v) in PBS. 

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... 

To detect structural variations in the O-PS structure of A. salmonicida from typical and atypical isolates we have used a microscale CE—MS-based method for analysis of A. salmonicida LPS directly on the bacterial cells. This method involves pretreatment ofbacterial cells ( 10 —10lu cells) with proteinase K, RNase and DNase followed by delipidation with mild acetic acid and subsequent MS analysis. Using this method, we have analyzed LPS structure of 39 typical and atypical isolates of A. salmonicida and related species (Table 1). All A salmonicida strains examined could be divided into three structural patterns, type A, type B, and type C, according to the structure of their corresponding O-PSs (Scheme 2). Majority of typical A. salmonicida isolates belonged to type A displayed the... 

After RNA digestion, it is necessary to digest the RNases by adding SDS to 0.5% and 100 p,g of proteinase K, incubate for 15 min at 37°C and extract with phenol/chloroform. Alternatively, RNases or proteinase K can be captured by StrataClean (after 2.5-fold dilution in water) or the ds RNA passed through an Immobilon filter (Section 3.1.2.7). The last method has the added benefit that ss probe RNA is also removed. RNA is then precipitated by adding 1 ml of ethanol as described in Table 3.1. 

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