In Vitro Transcription Translation Reactions, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Programmed ribosomal frameshifting generates a copper transporter and a copper chaperone from the same gene"
Article Title: Programmed ribosomal frameshifting generates a copper transporter and a copper chaperone from the same gene
Journal: Molecular cell
Figure Legend Snippet: Production of CopA(Z) depends on the integrity of the slippery sequence present in the copA gene (A) The slippery sequence CCCAAAG present in the copA gene at the end of the MBD1-coding segment. Slippage of the ribosome to the -1 frame would cause the ribosome to incorporate Gly-70 after Lys-69 and terminate translation at the following stop codon, generating the CopA(Z) protein. (B) Immunoblot of the lysates of E. coli cells carrying either the pCopA plasmid containing the wt copA gene, or pCopA-mSS in which the slippery sequence was mutated to prevent -1 PRF. The CopA (grey arrowhead) and CopA(Z) (black arrowhead) proteins were detected using anti-His 6 -tag antibodies. Proteins were fractionated in a 4-20% SDS gel. The SS-disrupting mutations (which do not change the sequence of the encoded protein) are underlined. (C) In vitro transcription-translation of a DNA template containing the first 94 codons of copA , followed by an engineered stop codon. The 16.5% Tris-Tricine SDS gel shows the [ 35 S]-labeled products corresponding to the complete 94 amino acid-long protein encoded in the 0 frame (grey arrowhead) or the 70 amino acid-long CopA(Z) produced via -1 PRF (black arrowhead). Disruption of SS in the template mSS by two synonymous mutations (shown in panel B) prevents production of CopA(Z) in vitro. .
Techniques Used: Sequencing, Plasmid Preparation, SDS-Gel, In Vitro, Labeling, Produced
2) Product Images from "Damaging the Integrated HIV Proviral DNA with TALENs"
Article Title: Damaging the Integrated HIV Proviral DNA with TALENs
Journal: PLoS ONE
Figure Legend Snippet: HT-TALENs and NS-TALENs cleave an HIV-1 DNA fragment in vitro . A. Schematic diagram representing HT-TALENs and NS-TALENs bound to their cognate DNA target sequence (thick lines). Relative locations of the Fok1 endonuclease, Flag epitope tag, and nuclear localization sequence (NLS) are indicated. Asterisks and grey boxes designate where a “NS” coding TALE repeat was used in the 5’ NS-TALEN construction. B. Western blot of in vitro transcription/translation reactions containing no expression plasmids, each TALEN alone, the HT-TALEN pair, or the NS-TALEN pair. C. Gel electrophoresis analysis of in vitro cleavage reactions containing no TALEN plasmids, the HT-TALEN pair, or the NS-TALEN pair. The HIV-1 target DNA fragment size is 747 bp, with expected on-target cleavage products of approximately 430 bp and 317 bp. Quantification of cleavage was performed using ImageJ software and is shown below the gel image. D. The HIV-1 target DNA fragment from (C) was mutated in the 5’ TALE binding site to create a set of triple mutant templates (Mut1-Mut4). The sequences of Mut1-Mut4 are depicted in bold, lowercase font and mutated positions are indicated by asterisks. Cleavage reactions containing either the HT-TALEN or NS-TALEN pairs incubated with the HIV-1 target templates were size fractionated by electrophoresis and quantified by densitometry with ImageJ [ 39 ].
Techniques Used: TALENs, In Vitro, Sequencing, FLAG-tag, Western Blot, Expressing, Nucleic Acid Electrophoresis, Software, Binding Assay, Mutagenesis, Incubation, Electrophoresis
3) Product Images from "Embryonic stem cell differentiation requires full length Chd1"
Article Title: Embryonic stem cell differentiation requires full length Chd1
Journal: Scientific Reports
Figure Legend Snippet: N-terminally truncated Chd1 is active in nucleosome assembly but lacks major phosphorylation sites. (a) DNA templates used for in vitro transcription/translation experiments. All sequences contain a Flag-tag at the end of exon 11. a , DNA fragment corresponding to the wt sequence including the natural transcription start site; b , DNA fragment corresponding to the transcript of the exon 2-deleted Chd1 locus; c-h , DNA fragments starting with in-frame ATGs located in downstream exons of the Chd1 gene. The red dot depicts the natural translation start codon. (b) Deletion of exon 2 results in protein translation starting at an ATG in exon 4. 35 S-labeled in vitro translation products were separated by SDS-PAGE and visualized by phosphoimaging. Stars depict Chd1 polypeptides used as size markers; arrows indicate translation products of templates a and b . (c) WT and N-terminal truncation mutants of Chd1 were expressed using the baculovirus system, purified and separated by gel electrophoresis and Coomassie Blue staining. (d) Mouse Chd1 catalyzes the assembly of nucleosomes on relaxed circular DNA in a dose dependent manner. Chromatin assembly reactions were carried out with increasing amounts (5, 10, 20 nM) of recombinant wt Chd1, and DNA supercoiling was analyzed after 70 min. (e) N-terminally truncated Chd1 is capable of efficient nucleosome assembly. Products of nucleosome assembly reactions with wt, Δ1-100 and Δ1-149 proteins (20 nM) were analyzed for DNA supercoiling efficiency. Reference lanes contain topoisomerase I-relaxed ( relaxed ) and supercoiled plasmid DNA ( supercoiled ), respectively. The positions of nicked (N) and fully supercoiled (SC) DNA species are indicated. (f) Chd1 is phosphorylated at the N-terminal SRR. Recombinant wt, Δ1-100 and Δ1-149 proteins were treated with alkaline phosphatase (AP), separated by SDS-PAGE and stained with silver. (g) Mouse Chd1 is a phosphoprotein in vivo . Treatment of protein extracts from Chd1 flox/flox cells with AP caused a mobility shift of Chd1 in SDS-PA gel electrophoresis, which was less pronounced for Chd1ΔSRR extracted from Chd1 Δ2/Δ2 cells. Chd1 was detected by immunoblotting with anti-Chd1 antibodies. Alpha-tubulin was used as a loading control.
Techniques Used: In Vitro, FLAG-tag, Sequencing, Labeling, SDS Page, Purification, Nucleic Acid Electrophoresis, Staining, Recombinant, Plasmid Preparation, In Vivo, Mobility Shift
4) Product Images from "Functional Analyses of the Three Simian Hemorrhagic Fever Virus Nonstructural Protein 1 Papain-Like Proteases"
Article Title: Functional Analyses of the Three Simian Hemorrhagic Fever Virus Nonstructural Protein 1 Papain-Like Proteases
Journal: Journal of Virology
Figure Legend Snippet: MassSpec analyses of selected polyprotein cleavage products. (A) nsp1α. (B) nsp1α+β. (C) nsp1α+β+γ. Products from in vitro transcription/translation reactions were separated on a 12% NuPAGE bis-tris gel
Techniques Used: In Vitro
5) Product Images from "Theiler's murine encephalomyelitis virus contrasts with encephalomyocarditis and foot-and-mouth disease viruses in its functional utilization of the StopGo non-standard translation mechanism"
Article Title: Theiler's murine encephalomyelitis virus contrasts with encephalomyocarditis and foot-and-mouth disease viruses in its functional utilization of the StopGo non-standard translation mechanism
Journal: The Journal of General Virology
Figure Legend Snippet: (a) Schematic representation of the wild-type and mutant TMEV and mengovirus region expressed by in vitro translation. (b) Coupled in vitro transcription–translation. Each plasmid (100 ng) in a 10 µl reaction volume was incubated at 30
Techniques Used: Mutagenesis, In Vitro, Plasmid Preparation, Incubation
Figure Legend Snippet: (a) 4–20 % SDS-PAGE of TMEV infected (m.o.i. 10) BHK-21 cell extracts from a pulse-labelling experiment. Lysates were prepared 6 h post-infection. (b) In vitro translation reactions of FMDV wild-type and its StopGo mutants.
Techniques Used: SDS Page, Infection, In Vitro
6) Product Images from "Crystallographic Analysis of Polypyrimidine Tract-Binding Protein-Raver1 Interactions Involved in Regulation of Alternative Splicing"
Article Title: Crystallographic Analysis of Polypyrimidine Tract-Binding Protein-Raver1 Interactions Involved in Regulation of Alternative Splicing
Journal: Structure(London, England:1993)
Figure Legend Snippet: Effect of Mutations of Raver1 PRIs on Binding to GST-PTB (A) Pull-down assays of binding of Raver1 PRI3 mutants to GST-PTB. Left: Loading controls for 35 S-Met-labeled PRI3-MS2 fusion proteins containing PRI3 wild-type (WT) and the mutants P6V, P6S, and P6A (see Experimental Procedures ). Right: Autoradiogram of PRI3-MS2 proteins pulled down with GST (1 μg) or GST-PTB (3 μg). (B) Pull-down assays of binding of Raver1 PRI4 mutants to GST-PTB. Left: Loading controls for 35 S-Met-labeled PRI4-MS2 fusion proteins. Right: GST pull-down of PRI4 constructs with GST (5 μg) or GST-PTB (15 μg). Although PRI4 constructs migrate as a doublet (as observed previously [ Rideau et al., 2006 ]), both products of the in vitro transcription-translation reaction contain the PRI since they bind PTB with the same affinity. (C) Comparison of binding of wild-type and mutant Raver1 PRIs to GST-PTB. Left: Loading controls. Right: GST pull-down of PRI4 constructs with GST (2 μg) or GST-PTB (6 μg). (D) PRI sequences from murine Raver1 (AAP33691), murine Raver2 (NP_898845), human matrin-3 (NP_001181884), and human hnRNP-L (NM_001533). Sequences shown for Raver1 are the 20 amino peptides used in pull-down assays; the shaded box indicates the sequences included in PRI-RRM2 chimeras for structural studies. Sequence similarity and identity within the PRI core are indicated. Residues in Raver1 PRI3 and PRI4 that were tested by mutagenesis are in boldface. See also Figure S3 .
Techniques Used: Binding Assay, Labeling, Construct, In Vitro, Mutagenesis, Sequencing