Lysis buffer, preparation

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. 

Preparation of eIF2from cell lysates for IEF 60 /(I of fast-flow Sepharose S (prewashed with lysis buffer) and 500 pg of cell lysate are mixed for 2 h at 4°. After centrifugation, the supernatant is removed and the beads are washed twice with lysis buffer containing 200 mM KC1. The eIF2 should be eluted with 50 pi of lysis buffer containing 400 mMKCl. 20 pi ofeluate is then mixed with 7 X sample buffer and urea (see previously), and loaded onto a prefocused IEF gel. 

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... 

Protocol for Preparation and Use of a Gram-Negative Bacteria Lysis Buffer... 

To prepare lysates from (nonstimulated) fibroblasts, cells from one confluent 78-cm2 plate are suspended in 0.15 ml lysis buffer (see below) and lysed by freezing and thawing six times and subsequent centrifugation at 13,000 x for 5 min. An aliquot of 0.05 ml of the supernatant is directly used for the enzyme assay. The preparation of tissue homogenate is described in section 6.1.4.1. GTP cyclohydrolase I, subheading Specimen . 

Blank reaction with cell lysate 10 pi of lysis buffer, 25 pi of freshly prepared 2 x reaction buffer, and 15 pi of water. Incubate at room temperature for 30 min in the dark, followed by the same oxidation procedure as for the reaction mixture. 

Pellet the cells by centrifugation at 4° C and freeze at-70°C or in a dry ice/ethanol bath. Prepare fresh lysis buffer by adding 1 mg/mL lysozyme, 2.5 U/mL Benzonase nuclease, 2 m. / MgCk 2 pL/mL protease inhibitor cocktail, and 1 mA/PMSF. Resuspend the frozen pellets in 500 pL fresh lysis buffer and shake on a plate shaker at 4°C for 30 min to lyse the cells. 

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. 

Preparation of rat heart tissue extracts. Lysis buffer containing 1% deionized Triton X-100 and 0.1% sodium azide in 50 mM Tris-HCl pH 7.5 was incubated with Chelex-100, for a minimum of 24 h at room temperature prior to use. Phenyl-methyl-sulfonyl-fluoride (PMSF) (final concentration 0.25 mM) was added to the buffer immediately prior to use. Rat hearts were frozen in liquid nitrogen, and stored at -SOX until analyzed. Lysis buffer was added to the homogenized tissue, the mixture was vortexed, sonicated for 1 min and incubated on ice for 30 min, with vortexing every 5-10 min. Aliquots were taken, centrifuged at 3,0(X) rpm for 15 min, and the supernatant analyzed for total protein and ferritin. Total protein was determined in the extracts by the BCA method (Pierce). 

Lysis buffer is used to prepare standards and phosphate buffer to prepare the spiking solutions. 

To prepare cell lysates from adherent cells, wash transfected adherent cells from Subheading 3.7 with ISOpl per well PBS using a multichannel pipette. Add 100pi lysis buffer per well, incubate for 10 min at RT, and then place the culture plate on ice. 

Lysis buffer (150mM NaCl, 50mM Tris, pH 8.5) (prepare 11) ... 

Prepare lx Passive Lysis Buffer with RPMI or Milli-Q water. 

To approximately 500pL of S. cerevisiae cell pellets on ice, add an equal volume of the fresh Lysis Buffer Solution (described in Section 13.2.2.2, prefiltered through a 0.22-pm membrane) containing protease inhibitors (follow the vendor s instructions for preparation) and vortex extensively to fully resuspend the pellet. 

The limited volume of the total cellular material from biopsy samples makes it necessary to use a slight modification of the lysis buffer recipe as compared with the cell culture lysis buffer. The following protocol describes a method for preparing whole cell lysates from LCM cells. The limited biopsy sample generally precludes the use of total protein assays before microarray printing. To compensate for this, one microarray slide is stained for total protein using a Sypro Ruby Protein blot stain (see Subheading 3.3.3.). 

Subtilisin-treated or nontreated HIV-1lav-i preparation was lysed with lysis buffer (50 mM Tris-HCl (pH 7.8) 80 mM KCl, 2.5 mM dithiothreitol,... 

Prepare assay blanks by adding 200 pi of lysis buffer instead of cell lysates to reaction tubes. 

Measure basal and Mn stimulated adenylyl cyclase activity from the same cells prepared for the cAMP mediated assay (from step 4 of Subheading 3.3.2). Transfer 600 pi of cells on ice to a 1-ml lysing syringe loaded with 600 pi of lysis buffer, and rapidly lyse the cells into a 12 x 75 mm glass tube on ice. 

To wash the GST-RBD beads, add 1 ml of lysis buffer and upside down five time, spin down the beads by centrifuging 2,000 X [ for 1 min. Prepare the beads in 100 pi lysis buffer containing 10 mg/ml BSA per sample. It is important to suspend well to aliquot equally. If you have ten samples to be tested, prepare the 100 pg beads in 1 ml lysis buffer containing 10 mg/ml BSA, then aliquot 100 pi each to new tube with O-cut tips. The final concentration of l-5 mg/ ml BSA must be added in the pulldown assay to avoid nonspecific binding of Ras proteins to the GST-RBD beads. 

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