Lysates, cell-free

Competition with release factors can be reduced by deactivating a release factor in a cell-free lysate prepared from a prokaryote. This option is not available in rabbit reticulyte lysate since mammals have only two release factors (RFs), termed eukaryotic release factors 1 and 3 (eRFl and eRF3). In eukaryotes, eRFl recognizes all three stop codons and eRF3 stimulates eRFl activity in the presence of GTP [41]. Deactivation of either RF would effectively deactivate all three stop codons, and at least one termination codon must function properly for the suppression method to be useful. 

The ELISA of cellular DNA fragmentation is a sandwich ELISA that measures apoptosis by quantitating the fragmentation and/or release of BrdU-labeled DNA. The commercial kit (Roche) can detect DNA fragments in the cell-free supernatants from cultured cells or cytoplasmic lysate of apoptotic cells prelabeled with BrdU... 

To obtain maximal protein productivity, it is necessary to construct an expression clone in which a protein coding region (open reading frame, mature region, domain, etc.) obtained from a cDNA of interest is inserted into the MCS of the pTD 1 vector. Typically, expression of the target protein at about 35-50 pg per mL of the translation reaction mixture can be obtained by using mRNA transcribed from the expression clone and the Transdirect insect cell kit. Furthermore, the expression clone can be effectively combined with other eukaryotic cell-free protein synthesis systems, such as rabbit reticulocyte lysate and wheat germ systems (tee Note 3). 

Additional information <1-7, 11, 14, 15, 17-19, 21, 30> (<7> cell-free synthesis in mRNA-dependent rabbit reticulocyte lysate system [40] <2,4,5> high activities in tissues where turnover of energy from adenine nucleotides is great, e. g. muscle [3] <1-6,11,14,15> tissue distribution [3,46] <2,5> rabbit and human carry a minimum of 2 sets of isozymes within an individual one set in muscle, erythrocytes, brain and another in liver, kidney and spleen [3]) [3, 40, 46]... 

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.

INITIAL PURIFICATION AND ASSAY OF ACTIVITIES IN CELL-FREE LYSATES... 

Fig. 10. Cell-free synthesis of t-PA glycoforms. The niRNA coding for t-PA was translated in a rabbit reticulocyte lysate in the presence of dog pancreas microsomes. Microsonies were isolated posttranslationally and the translocated, glycosylated products were separated by SDS-PAGE. Translation was carried out under conditions that either prevented (lane 2) or allowed (lane 3) proper folding of the t-PA molecule, yielding enzymatically active protein that was sensitive to natural inhibitors and stimulators.

In cell-free systems (recombinant GTPases or cell lysates), both toxins exhibit their enzymic effects only at a concentration 10-100 times higher than that applied to intact cells. This difference in concentration may be due to failure in toxin activation, which most likely occurs when the toxins enter the cell by receptor-mediated endocyto-sis. The same is true when the toxins are microinjected, thereby bypassing the entry (and activation) mechanism. 

Cell-free lysates prepared from 16 pathogenic and four non-pathogenic strains of pv. glycines were examined for the presence of plasmids by agarose gel electrophoresis. No plasmids were detected in the four non-pathogenic strains examined (Figure... 

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