in vitro transcription and translation protocols

... in vitro RNA synthesis. In vitro translation of a targeted message has been shown to be inhibited in the presence of antisense RNA in vitro (14), and in vivo translation has been blocked in ... polynucleotide kinase. 15. Incubate kinase reaction for 30 min at 37OC. 16. Heat inactivate the polynucleotide kinase by incubating the reaction at 70°C for 30 min. 17....

Ngày tải lên: 11/04/2014, 09:47

... translational level, the system of combined transcription ⁄ translation was decoupled. The in uence on the transcriptional level was investigated by in vitro transcription of the CAT gene using ... BcCSP. In vitro translation For determining the in uence of CSPs on the transcription apparatus the system for combined transcription ⁄ transla- tion was used without T7 RNA p...

Ngày tải lên: 07/03/2014, 21:20

... for 10 min. In vitro selection for mAb M2 binding was by adding 200-lL aliquots of the supernatant to the selection matrix and incubating at room temperature for 30 min. Unselected protein–plasmid ... the re-introduction of an initiator methionine codon and was achieved by PCR with primers incorporating the necessary sequence. The product was re-inserted into pMS401, and the resulti...

Ngày tải lên: 30/03/2014, 20:20

... USA) containing 10% heat-inactivated fetal bovine serum (Nuoding, China), and the HT-29 cells were grown in DMEM medium (GIBCO, USA) also containing 10% heat-inactivated fetal bovine serum. ... saline (PBS), and resuspended at 2 × 10 6 cells /mL in Annexin-V binding buffer. The supernatant (100 μL/tube) was incubated with 5 μL of Annexin-V-FITC (Invitrogen, USA) and 5 μL of...

Ngày tải lên: 26/10/2012, 08:57

... functional domain mapping of proteins where fragments that represent protein domains are expressed and functionally analyzed using in vitro assays. Furthermore, rapid in vitro protein–protein interaction studies ... with Analysis, sequencing and in vitro expression of PCR products 89 Analysis, sequencing and in vitro expression of PCR products 5.1 Introduction Analysis...

Ngày tải lên: 25/10/2013, 22:20

... in the mitochondrial apoptotic response induced by the toxin remains to be determined. Finally, we assign an important role to the endosomal acidic cathep- sins B and D in increasing the in vitro ... cathepsins B and D, with the major cleavages generated in vitro occurring mainly within domain III of ETA-A. Cell-free endosomes preloaded in vivo with ETA intraluminally proces...

Ngày tải lên: 18/02/2014, 04:20

... gentamicin (Gibco Invitrogen), 1% (v ⁄ v) l-glutamine, 5 lgÆmL )1 insulin (Gibco Invitrogen), 10 ngÆmL )1 epidermal growth factor (Gibco Invitrogen) and 240 lgÆmL )1 Geneticin Ò (G418; Gibco Invitrogen). ... (Gibco Invitrogen), 1% (v ⁄ v) l-glutamine (Gibco Invitrogen) and 1% (v ⁄ v) pyruvate (Gibco Invitrogen). Mouse mammary epi- thelial cells (HC11) were grown in RPMI 1640+ medium (Gi...

Ngày tải lên: 18/02/2014, 08:20

... ed-end actin and determine the level and distr ibution o f F -actin [34]. Therefore, it i s tempting to speculate that interactions between monomeric G-actin and actin-binding proteins are a ... fixed, permeabilized and stained for F-actin with rhodamine-conju- gated phalloidin. Th e effects of toxin B and latrunculin B on the actin filaments was examined by preincubating the cells wit...

Ngày tải lên: 19/02/2014, 16:20

... radioactivity incorporated into the nucleic acids was measured after TCA precipitation and plotted against the incubation time in minutes. T. Astier-Gin et al. Binding and replication of 3¢-end of HCV minus ... of the HCV minus-strand RNA is illustrated in Fig. 1. This fragment contains two domains: domains I and II. Structures of both domain I (A), as deter- mined by Schuster et al...

Ngày tải lên: 20/02/2014, 01:20