Neg035c001mc

Manufactured by New England Biolabs

The NEG035C001MC is a laboratory instrument for performing gel electrophoresis. It is designed to separate and analyze DNA, RNA, or protein samples based on their size and charge. The device features a high-performance power supply, adjustable voltage and current settings, and a clear acrylic cover for safe operation. Its compact design and easy-to-use interface make it a versatile tool for a variety of laboratory applications.

Automatically generated - may contain errors

Lab products found in correlation

L5020, by Promega (2 mentions) Illustra microspin g 25 column, by GE Healthcare (2 mentions) T4 polynucleotide kinase, by New England Biolabs (2 mentions) Yeast trna, by Thermo Fisher Scientific (1 mentions)

3 protocols using neg035c001mc

RNA-Protein Binding Assay with Drosha RBD

Check if the same lab product or an alternative is used in the 5 most similar protocols

100 pmol of WT or mutant RNA oligonucleotides (synthesized by Integrated DNA Technologies, Inc.) were 5’-end labeled with [γ³²P] ATP (Perkin Elmer, NEG035C001MC) and T4 polynucleotide kinase (New England Biolabs) as previously described (82 ). Unincorporated ATP was removed by Illustra MicroSpin G-25 Columns (GE Healthcare Life Sciences, UK). The radiolabeled RNA probe was denatured in buffer (50 mM Tris-Cl ; 100 mM KCl; 2.5 mM MgCl2; 100 mM NaCl) at 72°C, and then renatured gradually at a rate of 1℃/min. EMSA was performed by incubating the radiolabeled probe (100,000 cpm) with Drosha RBD protein, which was synthesized in vitro with a reticulocyte lysate system (Promega, L5020), for 2 h at room temperature in binding buffer (50 mM Tris-Cl, pH 7.5; 100 mM KCl; 2.5 mM MgCl2; 100 mM NaCl; 0.01% NP-40; 1 mM DTT; 5% glycerol; 10 μg/ml bovine serum albumin; 0.1mg/ml sperm DNA) (82 ). The RNA-protein mixtures were then electrophoresed in 8% acrylamide-TBE gels. Gels were dried and exposed to X-ray film for analysis. For the competition experiments, the proteins were incubated with labeled WT RNA and 50-fold molar excess of the unlabeled WT or mutated single-stranded RNA at room temperature for 2 hours before electrophoresis.

Jiang X., Prabhakar A., Van der Voorn S.M., Ghatpande P., Celona B., Venkataramanan S., Calviello L., Lin C., Wang W., Black B.L., Floor S.N., Lagna G, & Hata A. (2021). Control of ribosomal protein synthesis by the Microprocessor complex. Science signaling, 14(671), eabd2639.

+ Open protocol

+ Expand

RNA-Protein Interaction Assays

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

RNA EMSAs were performed as previously described (Kung et al., 2015 (link)), with minor modifications as described below. In vitro transcribed snoRNAs were 5’-end labeled with [γ−³²P] ATP (1 mCi; PerkinElmer, NEG035C001MC) and T4 polynucleotide kinase (NEB, M0201S) according to the manufacturer’s instructions and then pre-folded in Folding Buffer (10 mM Tris- HCl pH 7.4, 100 mM NaCl, 1 mM EDTA) by incubating for 2 minutes at 95°C, 5 minutes at 50°C, 15 minutes at 37°C, and 15 minutes at room temperature. After incubation, 50 mM KCl and 10 mM MgCl2 were added, and the RNA was incubated on ice for 10 minutes and stored on ice until used. Recombinant proteins of interest (e.g., PARP-1, DDX21) were incubated with folded 5’-end labeled RNA (10 nM) in Binding Buffer I [50 mM Tris-HCl pH 7.5, 100 mM KCl, 50 mM NaCl, 0.1% NP-40, 10 mM MgCl₂, 1 mg/mL yeast tRNA (ThermoFisher Scientific, AM7119), 1 mg/mL BSA, 5 % glycerol, 5 mM DTT, SUPERaseIn]. The samples were mixed gently, incubated for 15 minutes at room temperature, and subjected to polyacrylamide gel analysis as described below.

Kim D.S., Camacho C.V., Nagari A., Malladi V.S., Challa S, & Kraus W.L. (2019). Activation of PARP-1 by snoRNAs Controls Ribosome Biogenesis and Cell Growth via the RNA Helicase DDX21. Molecular cell, 75(6), 1270-1285.e14.

+ Open protocol

+ Expand

RNA-Drosha RBD Binding Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

100 pmol of WT or mutant RNA oligonucleotides (synthesized by Integrated DNA Technologies, Inc.) were 5'end labeled with [γ 32 P] ATP (Perkin Elmer, NEG035C001MC) and T4 polynucleotide kinase (New England Biolabs) as previously described (Celona et al., 2017) . Unincorporated ATP was removed by Illustra MicroSpin G-25 Columns (GE Healthcare Life Sciences, UK). The radiolabeled RNA probe was denatured in buffer (50 mM Tris-Cl ; 100 mM KCl; 2.5 mM MgCl2; 100 mM NaCl) at 72˙C, and then renatured gradually at a rate of 1℃/min. EMSA was performed by incubating the radiolabeled probe (100,000 cpm) with Drosha RBD protein, which was synthesized in vitro with reticulocyte lysate systems (Promega, L5020), for 2 h at room temperature in binding buffer (50 mM Tris-Cl, pH 7.5; 100 mM KCl; 2.5 mM MgCl2; 100 mM NaCl; 0.01% NP-40; 1 mM DTT; 5% glycerol; 10 μg/ml bovine serum albumin; 0.1mg/ml sperm DNA) (Celona et al., 2017) . The RNAprotein mixtures were then electrophoresed in 8% acrylamide-TBE gels. Gels were dried and exposed to X-ray film for analysis. For the competition experiments, the proteins were incubated with labeled WT RNA and 50fold molar excess of the unlabeled WT or mutated single-stranded RNA at room temperature for 2 hours before electrophoresis.

Jiang X., Prabhakar A., Voorn S.M.v.d., Ghatpande P., Celona B., Venkataramanan S., Calviello L., Lin C., Wang W., Black B.L., Floor S.N., Lagna G., & Hata A. (2020). Control of ribosomal protein synthesis by Microprocessor complex.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!