Proteinase solution preparation

Permeabilizmg solution Prepare O.lAf Tris-HCl, pH 8.0,50 mA/EDTA from stock solutions (IM and 0.5M respectively), diluting 1/10 with sterile distilled water. Just before use (Section 3.1., step 2), to 100 mL of this solution, prewarmed at 37°C, add 200 )iL proteinase K stock solution (final concentration 1 ig/mL). 

After 2 h incubation of the prepared antibody beads with UV-crosslinked extract in a cold room, the beads are washed 4 x with 100 /A RIPA buffer (50 mMTris-HCl pH 7.5, 150 rnMNaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) and lx with genomic DNA lysis buffer (50 mM Tris, pH 7.4, 10 mM EDTA, 500 mM NaCl, 2.5 mM DTT, 0.5 mM spermidine, 1% Triton X-100). Approximately 300 /(I of PK solution (1 mg/ml proteinase K in genomic DNA lysis buffer and 0.2 U//A RNase inhibitor) is added to the total lysate previously kept on ice and the beads are then incubated at 37° for 30 min. Gently flick the tubes to resuspend the beads every 10 min during the incubation. After removal of the proteinase K solution, 300 /A of RNA extraction solution (4 M guanidine thiocyanate, 0.5% sarkosyl, and 25 mM sodium citrate, pH7) is added to the beads, incubated for 10 min and the supernatant is mixed with 30 fig yeast tRNA (as a carrier) and 30 fil of 3 M sodium acetate. The RNA solution is phenol-chloroform extracted, ethanol-precipitated, and the pellet washed once with 70% ethanol. The dry pellet is used for 1st strand cDNA synthesis, followed by PCR analysis. The removal of proteins... 

For electrophoresis of the proteinase, saturated solutions of crude and purified preparations were made in 0.1 M Tris-glycine... 

Proteinase K is obtained from Beckman (Somerset, NJ). Other sources may be acceptable In using alternate sources, the occurrence of blue backgrounds between colonies, and oversize, blurry signals at colony sites after the final color development step indicates insufficient proteolytic activity. Prepare a solution of 200 pg/mL in IX SSC... 

Mix a proteinase-containing protein sample with 0.1 vol class- or enzyme-specific inhibitor solution. Include samples without inhibitor but with an appropriate amount of solvent used to prepare inhibitors, as well as known proteinases with their inhibitors as controls. 

Harvest the cells and prepare a cleared lysate as previously (4.3.4.), except that the volumes are increased to give approximately 40 ml cleared lysate. Adjust to 0.1 M NaCl, 0.2% Sarkosyl, and add 4 mg pancreatic RNAase (from a 5 mg/ml stock solution in 0.1 M sodium acetate, pH 5.5, previously boiled for 10 min.). Incubate for 1 hour at 37°C. Add 1.0 ml of a 1 mg/ml solution of proteinase K (BDH Biochemicals) and continue incubation for a further hour at 55°C. 

Mouse tissue (mouse tail) lysis buffer (2X) 8 M urea, 20 mM EDTA (prepared from a stock solution of 0.5 M, pH 8.0, 1% N-lauroylsarcosine (Sigma-Aldrich, Cat. No. L-9150) (make stock solution, 30%), 0.2 M Tris-Cl, and 0.4 M NaCl. The 2X lysis buffer is diluted to IX with distilled water. Proteinase K is added to 100 pg/mL when needed. 

Enzymatic Action. Proteinases (also called proteases) present in crude or unpurified protein preparations often catalyze the breakdown of proteins by hydrolyzing the peptide bonds. Since these enzymes act more slowly at low temperatures, crude protein solutions are frequently kept cold (0 to 2°C) during the early stages of purification. In some cases it is also necessary to add inhibitors of proteinases, such as p-methylphenylsulfonyl fluoride (PMSF). 

To isolate genomic DNA from E. coli, the cells are treated with lysozyme and then lysed by SDS in the presence of proteinase K. Proteinase K, which is active even in SDS solution, degrades proteins including nudeases. Cell debris, polysaccharides and unhydrolysed protein are removed by precipitation at room temperature with cetyltrimethylammonium bromide (CTAB). DNA is isolated from the supernatant by precipitation with alcohol. RNA can be removed from DNA preparations by incubation with DNase-free RNase. Further purification can be effected by a phenol/ chloroform/isoamyl alcohol (25 24 1) extraction, and/or by CsCl gradient centrifugation (see Sect. 4.3.4.2 ) to remove the remaining protein and RNA. 

Proteinase K A stock solution of proteinase K (Sigma, Poole, Dorset, UK P2308) is prepared by dissolving 10 mg of enzyme in 1 mL of water, to give a 10 mg/mL concentration. The enzyme is aliquoted and stored frozen. The aliquots should not be thawed and re-frozen more than twice. For use, 5 mL of the stock solution is diluted into 5 mL of sterile 50 mM Tris-HCl, pH 8.0, 1 mM ethylenediam-inetetraacetic acid (EDTA). 

We prepared two samples, each containing an aqueous suspension (0.5 ml) of micro-capsules with urease (7.4 x 10 capsules/ml) in 0.01 M Tris-HCl (pH 8.0). A solution (0.5 ml) of proteinase K at 4 mg/ml in the same buffer was added to the first sample, and the same amount of the buffer was added to the second sample. Then, the solutions were incubated at 37°C. Every 10 min, 50-ml portions were taken and added to the urease assay mixture (1.95 ml) with 125 mM urea. Free urease at 20 pg/ml instead of microcapsules was used as a reference. 

Lysis buffer Prepare immediately before use. Add 250 /il of 2x stock solution, 10 /il of proteinase K, and 2.5 /il of tRNA, and bring the volume to 500 /il with DEPC water. 

In this chapter, we review the enzymatic degradation mechanisms of PLA prepared from melt-crystallized, solvent-cast, and blend films, mainly by using proteinase K enzyme molecules. The degradation mechanisms of thin films with thickness of about lOOnm and solution-grown lamellar crystals are also discussed. 

To study the enzymatic degradation of the free amorphous region, completely amorphous PLLA thin film was prepared (see Section 22.3.2), and the erosion rate was directly monitored by using QCM in the nanogram per square centimeter regime [74]. The enzymatic erosion rate was dependent on the concentration of proteinase K, and the thin amorphous film of lOOnm thickness was completely hydrolyzed in 20 min when the concentration of the enzyme was >100 qg/mL. During the course of enzymatic degradation, even if the enzyme solution was replaced with a buffer solution (i.e.. 

Store at room temperature for up to 3 months, or at -20"C. If stored at -20 C, heating is necessary to redissolve the SDS. Prepare a stock solution of proteinase K (2.5 mg/ml USB 20818) in TE. Store at -20°C. The solution can be ffeeze-thawed multiple times. Just before use, combine transcription stop and proteinase K in a 20 1 (v/v) ratio before adding to samples. 

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