Rabbit Reticulocyte Lysate (RRL, Promega L416A) or Transdirect Insect Cell Extract (Shimadzu 292-30000-91) from Spodoptera frugiperda ovary Sf21 cells was used, in accordance with each manufacturer's protocol. The final reaction concentrations for potassium, magnesium, and template RNA were adjusted to 150 or 100 mM, 1.1 mM, and 0.03 µg/µL (RRL) or 0.3 µg/µL (Sf21), respectively. Products were labeled with L-[35S]-methionine (Perkin-Elmer, NEG709A) and separated by SDS-PAGE, as described previously (Kamoshita et al. 1997 (link)). Radioactivities of polypeptides on the images scanned with a Typhoon 9410 Imager (GE Healthcare) were measured as volume values V of photo-stimulated luminescence (PSL) in ImageQuant TL software (GE Healthcare). When polypeptides with different lengths were compared, the PSL value was divided by the number of methionine residues in each polypeptide. Readthrough efficiency of certain mRNA was calculated as (VRT/nRT)/(VRT/nRT + VRLuc/nRLuc), where VX and nX represent the PSL V value and the number of methionine residues, respectively, for translated polypeptide X, where polypeptide X is either RT (stop codon readthrough) or RLuc (Renilla luciferase). IRES activity was calculated as (VFLuc/nFLuc)/(VRLuc/nRLuc).
Neg709a
Manufactured by PerkinElmer
Sourced in United States
The NEG709A is a laboratory equipment product from PerkinElmer. It is a compact and versatile device designed for various scientific applications. The core function of the NEG709A is to perform high-precision measurements and analyses within a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
His pur 88222, by Bio-Rad (2 mentions)
L1170, by Promega (2 mentions)
Imagequant tl, by GE Healthcare (1 mentions)
Typhoon 9410 imager, by GE Healthcare (1 mentions)
Tnt t7 coupled reticulocyte lysate system, by Promega (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Tnt sp6 coupled reticulocyte lysate system, by Promega (1 mentions)
D0422, by Merck Group (1 mentions)
Sephadex protein a g beads, by Merck Group (1 mentions)
Anti myc positive control antibody, by Cell Signaling Technology (1 mentions)
Nap 5 column, by GE Healthcare (1 mentions)
10 protocols using neg709a
1
Cell-Free Translation Assay Protocol
2
In Vitro Protein Synthesis and Interaction
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35S-labeled proteins (TBX5, NKX2-5, RBPjk, NICD, BAF60c serial deletions, Myocardin serial deletions) (Wang et al., 2001 (link)) were synthesized with the TnT SP6 coupled reticulocyte lysate system (Promega, L4600) or TnT T7 coupled reticulocyte lysate system (Promega L4610) and labeled with 35S methionine (Perkin Elmer NEG709A). 5 µl of each synthesized protein was analyzed with SDS-PAGE gel and exposed to X-ray film for evaluation. GST-BAF60c, GST-RBPjk, GST-TBX5 and GST were expressed in E. coli strain BL21 and purified with glutathione Sepharose 4B (GE Healthcare, 17-0756-01). The beads were incubated with 35S-labeled target proteins overnight at 4°C and washed with PBST for three times. The beads were then boiled in loading buffer. The protein was analyzed with SDS-PAGE gel and autoradiography.
3
Radiolabeling of Newly Synthesized Proteins
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Wild-type or H19-overexpressing GH3 cells were washed with phosphate-buffered saline (PBS) and incubated in methionine and cysteine-free DMEM (Sigma, D0422) for 30 min. The 35S-labelled l -methionine and l -cysteine mix (75 μCi in a 35-mm dish) (PerkinElmer, NEG709A) was then added to the medium, and the cells were incubated for an additional 30 min. The cells were quickly washed with cold PBS and lysed in EBC buffer. Proteins were resolved by SDS-PAGE, and newly synthesized proteins were detected by autoradiography.
4
In Vitro Transcription and Translation of Recombinant Proteins
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PCR products were generated using 3XFlag-CRP (Extended Data Table 1 ) as template with reverse primer either containing a stop codon (3XFlag-CRPStop), or lacking a stop codon but with a 30 nucleotide polyT extension (3XFlag-CRPNSKn). PCR products were transcribed and translated in vitro using TnT T7 Quick rabbit reticulocyte lysate (Promega, # L5540) in the presence of radioactive methionine (Perkin Elmer, #NEG709A). Reactions were typically for 30 mins at 30°C, at which time recombinant proteins were added for another 10 mins. Aliquots were resolved by SDS-PAGE, and dried gels were either exposed to film or to a PhosphorImager screen.
5
Protein Expression Analysis by SDS-PAGE
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Cells were cultured in 35 mm dishes. Before finishing each experimental condition, cells were labeled with 20 µCi of 35S-methionine (PerkinElmer, NEG709A, Waltham, MA, USA) for 1 h. Cells were washed with ice-cold 0.1 M phosphate-buffered saline (PBS) and scrapped with lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 1% sodium dodecyl sulfate (SDS)) containing 2 mg/mL of protease inhibitor cocktail (cOmplete, Roche, 11836145001 Basel, Switzerland) 30 µg of protein was separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a PVDF membrane, and analyzed with a Typhoon 8600 phosphorimaging device (GE Healthcare, Chicago, IL, USA).
6
In Vitro Transcription and Translation of Recombinant Proteins
7
Purification of Radiolabeled Recombinant Proteins
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Coupled transcription/translation reactions were carried out per the manufacturer’s instructions (L1170; Promega). Briefly, rabbit reticulocyte lysate was incubated with 1 μg of the appropriate expression plasmid and 2 ml of [35S]l -methionine (1,000 Ci/mMol at 10 mCi/ml) (NEG709A; Perkin Elmer) for 90 min at room temperature. Reaction mixtures were diluted in 1 ml of 50 mM Tris-HCl (pH 7.5), 10 mM NaCl, and 1 mM dithiothreitol (DTT). A volume of 100 μl of DEAE-Sepharose (DFF100; Millipore Sigma) slurry was added to the reaction mixtures and incubated on a nutator for 30 min at room temperature. The slurry was washed three times with 1 ml of 50 mM Tris-HCl (pH 7.5), 10 mM NaCl, and 1 mM DTT and eluted with 500 μl of 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 1 mM DTT. Eluents were collected and incubated with 50 μl of packed nickel-nitrilotriacetic acid resin (His-Pur 88222; Bio-Rad) at room temperature with nutating for 30 min. The beads were washed three times with 1 ml of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 20 mM imidazole, and 1 mM DTT. Proteins were eluted using 100 μl of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 300 mM imidazole, and 1 mM DTT.
8
IFN-α2 Autoantibody Immunoprecipitation Assay
9
Forebrain neuroepithelial cell protein synthesis
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E8.5 and E10.5 forebrain neuroepithelium was dissected as described (Chau et al., 2015 (link)) and trypsinized. Cells were serum starved in methionine-free DMEM for 1 hr at 37°C, then incubated with 51 μCi 35S-Methionine (Perkin Elmer NEG709A) at 37°C for an additional hour. Cycloheximide (50 μg/ml) was added to stop translation. 35S-Methionine incorporation was measured using scintillation counter.
10
Purification of radiolabeled proteins
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Coupled transcription/translation reactions were carried out per manufacturer's instructions (Promega, L1170). Briefly, rabbit reticulocyte lysate was incubated with 1µg (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.10.420794 doi: bioRxiv preprint of the appropriate expression plasmid and 2mL of [ 35 S] L-methionine (1,000 Ci/mMol at 10mCi/mL) (Perkin Elmer, NEG709A) for 90 minutes at room temperature. Reactions were diluted in 1mL of 50mM Tris-HCl (pH=7.5), 10mM NaCl, and 1mM DTT. 100µL of DEAE-Sepharose (Millipore Sigma, DFF100) slurry was added to the reactions and incubated on a nutator for 30 min at room temperature. The slurry was washed three times with 1mL of 50mM Tris-HCl (pH=7.5), 10mM NaCl, and 1mM DTT, and eluted with 500µL of 50mM Tris-HCl (pH=7.5), 500mM NaCl, and 1mM DTT. Eluents were collected and incubated with 50µL of packed Ni-NTA resin (Bio-Rad His-Pur 88222) at room temperature with nutating for 30 min. The beads were washed three times with 1mL of 50mM Tris-HCl (pH=7.5), 150mM NaCl, 20mM imidazole and 1mM DTT.
Proteins were eluted using 100µL of 50mM Tris-HCl (pH=7.5), 150mM NaCl, 300mM imidazole and 1mM DTT.
The copyright holder for this preprint this version posted December 11, 2020. ; https://doi.org/10.1101/2020.12.10.420794 doi: bioRxiv preprint of the appropriate expression plasmid and 2mL of [ 35 S] L-methionine (1,000 Ci/mMol at 10mCi/mL) (Perkin Elmer, NEG709A) for 90 minutes at room temperature. Reactions were diluted in 1mL of 50mM Tris-HCl (pH=7.5), 10mM NaCl, and 1mM DTT. 100µL of DEAE-Sepharose (Millipore Sigma, DFF100) slurry was added to the reactions and incubated on a nutator for 30 min at room temperature. The slurry was washed three times with 1mL of 50mM Tris-HCl (pH=7.5), 10mM NaCl, and 1mM DTT, and eluted with 500µL of 50mM Tris-HCl (pH=7.5), 500mM NaCl, and 1mM DTT. Eluents were collected and incubated with 50µL of packed Ni-NTA resin (Bio-Rad His-Pur 88222) at room temperature with nutating for 30 min. The beads were washed three times with 1mL of 50mM Tris-HCl (pH=7.5), 150mM NaCl, 20mM imidazole and 1mM DTT.
Proteins were eluted using 100µL of 50mM Tris-HCl (pH=7.5), 150mM NaCl, 300mM imidazole and 1mM DTT.
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