Proteinase buffer solution preparation

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... 

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.. 

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. 

X SSC buffer 3MSodium chloride, 0.3Msodium citrate, pH at usted to 7 with sodium hydroxide. Sterilize by autodaving, store at room temperature. Proteinase K is obtained from Beckman, Somerset, hQ. 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. 

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. 

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