Lysate buffer, solution preparation

Figure 5.6 Overview of steps involved in the preparation of a cell-free lysate. The cells are resuspended in a buffered solution at a specified cell density. To this suspension is added a cocktail containing several proteolytic inhibitors. The cells in the suspension are lysed (here by homogenization). Finally the lysate is subjected to a very low speed centrifugation such as 5000g for 10 minutes to remove unbroken cells.

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... 

Reaction mixture 25 pi of freshly prepared 2 x reaction buffer, 15 pi of water, and 10 pi of filtered cell or tissue lysate (total volume of 50 pi). The reaction mixture is incubated for 30 min at 37°C in the dark, followed by adding 10 pi of oxidation solution. After oxidation for 30 min in the dark at room temperature, 10 pi of 1% ascorbic acid is added, mixed, and centrifuged for 20 min at 14,000 xg through a Micron 10,000 filter (Millipore, Ultracel YM-10). The filtrate is analyzed by HPLC (ideally only 20 pi of a 1 2 dilution with water are injected into the HPLC system). The starting lysate of 10 pi was diluted sevenfold. 

Discontinuous iodixanol step gradients are formed in quick seal tubes (25 x 89 mm, Beckman) by underlaying and displacing the less dense cell lysate (15 ml) with iodixanol (5,5 [(2-hydroxy-1-3-propanediyl)-bis(acetylamino)] bis [N,Ar,-bis(2,3dihydroxypro-pyl-2,4,6-triiodo-l,3-benzenecarboxamide]) prepared using a 60% (w/v) sterile solution of OptiPrep (Nycomed) and PBS-MK buffer (1 x PBS containing 1 mM MgCl2 and 2.5 mM KC1). Therefore,... 

The reaction mixture was prepared by mixing 800 fiL of substrate solution containing 0.01 M p-nitrocatechol sulfate (dipotassium salt), 1.71 M NaCl, and 0.5 M acetate buffer (pH 5.0) with 200 fiL of either leukocyte and platelet lysate or saliva. After incubation for an hour at 37°C, a 50 fiL aliquot was mixed with 100 fiL of bovine serum albumin (13 mg/mL) and 1800 fiL of cold 95% ethanol. After centrifugation to remove proteins, 20 /iL was injected into the HPLC system. The reaction was linear with up to 200 nL of lysate. 

Plasma-free platelet solution (washed platelet, WP) and PRP were prepared by adding the platelet pellets to PBS and PPP, respectively. 0,6 ml of WP or PRP was placed on each of the hydrogel films of 1.8 cm in a vial and allowed to stand for 1 hr at 37 °C. Then, the films were vigorously washed with PBS and put into 2 ml of 0.1 M phosphate buffer (PB) containing 0.5 % Triton-XlOO to lyse the adhered platelets. Lactic acid dehydrogenase (LDH) activity of the lysate was determined with an enzymatic method to count the adhered platelets with the use of a calibration curve of platelet counts(Tamada,Y., Kyoto University, Doctor Thesis, 1989.). To confirm the reproducibility, the platelet adhesion experiment was repeated five times for the same film. A Silastic film, donated by Dow Corning Cor., Ltd., Midland, USA, was used as a reference material. 

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