All enzymes as well as plasmid pGEM1, the TNT SP6 Quick Coupled System and rabbit reticulocyte lysate were from Promega (Madison, WI). ER rough microsomes from dog pancreas were from tRNA Probes (College Station, TX). [35S]Met were from Perkin Elmer. The restriction enzymes were purchased from Roche Molecular Biochemicals. The DNA purification kits were from Thermo (Ulm, Germany). All the oligonucleotides were purchased from Sigma-Aldrich (Switzerland).
Tnt sp6 quick coupled system
Manufactured by Promega
Sourced in United States
The TNT SP6 Quick Coupled System is a cell-free, in vitro transcription and translation system that allows for the rapid synthesis of proteins from DNA templates. It combines the SP6 RNA polymerase and rabbit reticulocyte lysate components in a single reaction mixture to facilitate the efficient expression of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit reticulocyte lysate, by Promega (1 mentions)
Dna purification kit, by Thermo Fisher Scientific (1 mentions)
35s met, by PerkinElmer (1 mentions)
Fla 3000 phosphoimager, by Fujifilm (1 mentions)
Fla3000 phosphorimager, by Fujifilm (1 mentions)
Endo h, by New England Biolabs (1 mentions)
35s methionine, by PerkinElmer (1 mentions)
6 protocols using tnt sp6 quick coupled system
1
In Vitro Protein Synthesis Assay
2
In Vitro Protein Synthesis Assay
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All constructs were transcribed and translated in the TNT SP6 Quick Coupled System (Promega Biotech AB, Madison, WI). 100 ng DNA was mixed with a master mix consisting of 0.5 µl 35S-methionine (5 µCi) (PerkinElmer, Boston, MA). In positive samples, 0.5 µl column-washed canine pancreas rough microsomes (tRNAprobes, College Station, TX) were added. All samples were incubated at 30 °C for 90 min.
3
In Vitro Translation of Membrane Proteins
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Constructs in pGEM1 were transcribed and translated in the TNT SP6 Quick Coupled System (Promega). 75 ng DNA template, 0.5 μL 35S-Met (5 μCi) and 0.25 μL microsomes (tRNA Probes) were added at the start of the reaction, and samples were incubated for 90 min at 30 °C. Translation products were diluted in 50 μL of loading buffer and analyzed by SDS–PAGE. The gels were quantified using a Fuji FLA-3000 phosphoimager and Image Reader 8.1j software. The membrane-insertion probability of a given TM sequence was calculated as the quotient between the intensity of the singly glycosylated band divided by the summed intensities of the singly glycosylated and doubly glycosylated bands.
4
In Vitro Expression of Engineered Mgst2
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Wild-type Mgst2 gene was engineered by introducing N-linked glycosylation site (Asn-Ser-Thr) followed by a linker sequence (Gly-Ser-Ala-Gly-Ser-Ala-Gly-Ser-Ala-Gly) between the residues 2Ala-3Gly. The engineered Mgst2 gene was cloned into pGEM1 vector for expression. The construct was transcribed and translated in vitro using TNT® SP6 Quick Coupled System (Promega) in the presence and absence of column-washed rough microsomes (CRM) of pancreas from dog. In all, 5 μl of reticulocyte lysate, 500 ng of plasmid DNA, 0.5 μl of [35S]Met, and 0.5 μl of CRM were mixed and incubated at 37 °C for 90 min. For Endo H treatment, 9 μl of the TNT reaction was mixed with 1 μl of 10× glycoprotein denaturing buffer, 0.5 μl of Endo H (500,000 units/ml; New England BioLabs), 7.5 μl of MillQ water, and 2 μl of 10× GlycoBuffer 3. The reaction mix was incubated at 37 °C for 1 h. Translated products were analyzed by 12% SDS-PAGE after heating at 40 °C for 10 min, and the gel was visualized on Fuji FLA-3000 PhosphorImager (Fuji film) with the Image Reader 8.1J/Image Gauge software.
5
Radiolabeled Mitochondrial Protein Import
6
Membrane Insertion Probability Assay
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Variants in pGEM1 were transcribed and translated in one step using
the TNT SP6 Quick Coupled System (Promega, USA). The reaction mixtures
contained 75 ng of DNA template, 0.5 μL of EasyTag (5 μCi), and 0.25 μL of column-washed canine microsomes (tRNA
Probes, USA) and were incubated for 30 min at 30 °C. Translation
products were subsequently ultracentrifuged (100 000g for 15 min) on a sucrose cushion and analyzed by SDS-PAGE.
The protein bands were quantified using a Fuji FLA-3000 phosphoimager
and Image Reader 8.1j software. As mentioned above, the membrane-insertion
probability for a given pHLIP-derived segment was calculated as the
quotient of the intensity of the double glycosylated band divided
by the summed intensities of the singly glycosylated and doubly glycosylated
bands. For the proteinase K (PK) protection assay, 2 μL of proteinase
K (2 mg/mL) was added to the sample, and the digestion reaction was
incubated for 15 min on ice. Before SDS-PAGE analysis, the reaction
was stopped by adding 2 mM phenylmethanesulfonyl fluoride.41 (link)
the TNT SP6 Quick Coupled System (Promega, USA). The reaction mixtures
contained 75 ng of DNA template, 0.5 μL of EasyTag (5 μCi), and 0.25 μL of column-washed canine microsomes (tRNA
Probes, USA) and were incubated for 30 min at 30 °C. Translation
products were subsequently ultracentrifuged (100 000g for 15 min) on a sucrose cushion and analyzed by SDS-PAGE.
The protein bands were quantified using a Fuji FLA-3000 phosphoimager
and Image Reader 8.1j software. As mentioned above, the membrane-insertion
probability for a given pHLIP-derived segment was calculated as the
quotient of the intensity of the double glycosylated band divided
by the summed intensities of the singly glycosylated and doubly glycosylated
bands. For the proteinase K (PK) protection assay, 2 μL of proteinase
K (2 mg/mL) was added to the sample, and the digestion reaction was
incubated for 15 min on ice. Before SDS-PAGE analysis, the reaction
was stopped by adding 2 mM phenylmethanesulfonyl fluoride.41 (link)
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