To crosslink protein-RNA complexes, cells cultured in a 10 cm dish were washed with PBS twice and received 254 nm UV irradiation (1500 J/m2) before protein extraction. Cells were dispersed in 10 mM HEPES (pH 7.6), 15 mM KCl, 2 mM MgCl2, and 0.1 mM EDTA with protease inhibitors (G6521; Promega). NP-40 was then added to give a final concentration of 0.2%. After initial centrifugation (600 × g for 1 min), the supernatant was further centrifuged (21,500 × g for 15 min) to obtain nuclear fraction. Nuclear proteins were extracted with RIPA buffer [50 mM HEPES (pH 7.6), 150 mM NaCl, 0.5% Triton X-100, and 0.5% sodium cholate] with protease inhibitors (G6521; Promega) and RNase OUT (Thermo). An antibody preincubated with protein G magnetic beads (Thermo) was added to the extract in the presence of RQ1 RNase-Free DNase (Promega) and incubated for 1 h at 4°C. The magnetic beads were washed with Tris-buffered saline + Tween-2 (TBST) four times and further washed with TBST + 1 M NaCl. Then, the beads were incubated with protease K (1 h at 37 °C). Then, phenol-chloroform extraction and ethanol precipitation were performed.
G6521
Manufactured by Promega
Sourced in China
The G6521 is a spectrophotometer designed for measuring the absorbance of samples in a variety of applications. It provides accurate and reliable measurements of sample concentration and purity.
Automatically generated - may contain errors
Lab products found in correlation
Rq1 rnase free dnase, by Promega (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Anti dykddddk tag antibody magnetic beads, by Fujifilm (1 mentions)
Anti rabbit igg haul, by Proteintech (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Sa00001 2, by Proteintech (1 mentions)
Ripa lysate, by Solarbio (1 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
Protein g magnetic beads, by Thermo Fisher Scientific (1 mentions)
4 protocols using g6521
1
UV-Crosslinking of Protein-RNA Complexes
2
Crosslink Protein-RNA Complexes and Analyze
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To crosslink protein-RNA complexes, cells cultured in a 10 cm dish were washed with PBS twice and received 254 nm UV irradiation (1500 J/m2) before protein extraction. Cellular proteins were extracted with RIPA buffer [50 mM HEPES (pH 7.6), 150 mM NaCl, 0.5% Triton X-100, and 0.5% sodium cholate] with protease inhibitors (G6521; Promega). Anti-DYKDDDDK tag antibody magnetic beads (Fujifilm Wako Pure Chemical) were added to the lysate and incubated for 1 h at 4 °C. The magnetic beads were washed with TBST four times and further washed with TBST + 1 M NaCl. For western blotting analysis, washed beads were mixed with sodium dodecyl sulfate-containing sample buffer and heated at 95 °C. To retrieve RNA, beads were treated with RQ1 RNase-Free DNase (Promega), and after a TBST wash, they were incubated with protease K (1 h at 37 °C). Then, phenol-chloroform extraction and ethanol precipitation were performed.
3
RNA Extraction from Cell Fractions
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Cells were dispersed in 10 mM HEPES (pH 7.6), 15 mM KCl, 2 mM MgCl2, and 0.1 mM EDTA with protease inhibitors (G6521; Promega), and NP-40 was added to give a final concentration of 0.2%. After initial centrifugation (600 × g for 1 min), the supernatant was further centrifuged (21,500 × g for 15 min), and an RNA-containing pellet was obtained by phenol-chloroform extraction and ethanol precipitation. After 600 × g centrifugation, the precipitating nuclear fraction was mixed with Trizol (Thermo) to isolate RNA.
4
Western Blot Analysis of ENO1 Protein Expression
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Following 48 h of transfection, 200 µL of lysate was added to the six-well plate (RIPA lysate: R0010; Solarbio, Beijing, China; PMSF: P0100; Solarbio, Beijing, China; protease inhibitor: G6521, Promega, Madison, WI, USA; using a 100:1:1 ratio). The cell protein lysate was mixed with protein sample loading buffer (LT103; Epizyme, Shanghai, China) at a 4:1 ratio, and then boiled for 5 min at 95 °C in a metal bath. An identical volume of (25 µg) protein was added to the 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis plate (SDS-PAGE) for electrophoresis. The protein was then transferred to the polyvinylidene fluoride membrane. The membrane was sealed with 5% bovine serum albumin for 2 h and then incubated with corresponding primary antibodies at 4 °C overnight. The sample was rinsed with TBST the next day and then incubated with the corresponding secondary antibody at ambient temperature for 2 h. Chemiluminescence was displayed using Fluorescent XRS+ (Bio-Rad, Hercules, CA, USA). β-actin served as the internal reference, the first antibody was anti-ENO1 (1:1000, ET1705-56; Huabio, Hangzhou, China) and anti- β actin (1:10000; 81115-1-RR; Proteintech, Wuhan, China). The second antibody was Anti-Rabbit IgG (Haul) (1:10000, SA00001-2; Proteintech, Wuhan, China)
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