Lysate preparation from cell culture

The level of enzyme needed can influence the choice of preparation used for the study. Microsomal preparations from cell cultures allow the use of higher concentrations of active enzyme per unit volume than use of whole cells or cell lysates. The use of whole, viable cells allows the use of longer incubation times but at a lower enzyme concentration per unit volume. In addition, adequate oxygen transfer and nutrient concentrations are needed to maintain culture viability. These requirements impose limitations on cell concentration. In addition, microsomes cannot be efficiently prepared from all cultured cell types. We have found that standard microsome preparation procedures as used for human or rodent liver were unsuitable for isolating active enzymes from human lymphoblasts, and this appears to be a general property of cultured cell lines. Specific catalytic activities in microsomes were lower than for whole cell lysates. This loss of activity appears to happen in other mammalian cell systems which has led to the common use of whole cell lysates.With human lymphoblasts, shortening the length of... 

The methods described herein encompass (1) preparation of a whole cell lysate from either cell culture or tissue samples, (2) protein lysate microarray printing, (3) immunostaining, and (4) microarray spot analysis. 

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.). 

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. 

In insect cells, yeast or mammalian cells, microsomal preparations can be prepared, however, the yield of microsomal protein from cultured cells is often low. Sonication of a frozen whole cell lysate appears to provide a fully activated preparation and is easier than preparing microsomes. If intact microsomes are needed from transfected mammalian cells, a recent procedure delineated by Sukodhub and Burchell is recommended for their preparation (Sukodhub, 2005). 

Lynch et al. (47) made use of such an antiserum to isolate vWf mRNA from polysomes of cultured endothelial cells. From the enriched mRNA, single-stranded cDNA probes were derived. A cDNA library made from endothelial cell mRNA larger than 28S rRNA was screened with this probe. Nine clones out of 3000 were positive and appeared to share a common region. The longest clone of 2.4 kbp hybridized to an endothelial cell mRNA species that was about 9.5 kb in size. When mRNA from this partial cDNA clone was prepared with the SP6 RNA polymerase system and translated in a rabbit reticulocyte lysate, several polypeptides were produced that reacted with a specific polyclonal rabbit antibody against vWf, and also with four out of a collection of 30 monoclonal antibodies raised against vWf. Final proof that the cDNA clone isolated by Lynch et al. was an authentic vWF cDNA clone was obtained by comparing the sequence of the 3 end of the clone with the sequence of the carboxy-terminal 10 amino acids (270). 

Figure 2, Trp-P-1 activated caspase-3-like proteases, MNCs were cultured with or without 10 pM Trp-P-1 for 3 h. Whole cell lysate was prepared and subjected to the assay for caspase-1- and -3-like protease activities using their peptide substrates, Ac-YVAD-MCA and Ac-DEVD-MCA, respectively, as described in Materials and Methods, Caspase activity was calculated as the amounts ofAMC released from substrates per min. Data represent the means S,D. of triplicate experiments. Asterisks indicated significant difference from corresponding control (Student s t-test, P < 0.05).

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