Cells lysate was prepared as described previously32 (link). Lysate containing 15 μg total RNA was loaded on to 10-50% linear sucrose gradients containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 μg/ml cycloheximide, and 2 U/ml SUPERase In RNase Inhibitor and sedimented by ultracentrifugation at 36,000 rpm for 2 hr at 4 °C with SW41 rotor (Beckman Coulter). Gradients were fractionated using Gradient station (Biocomp). UV absorbance was detected by ECONO UV monitor (Biorad).
Econo uv monitor
Manufactured by Bio-Rad
Sourced in Canada, United States
The Econo UV Monitor is a compact and efficient device designed to continuously monitor the ultraviolet (UV) absorbance of liquids flowing through a chromatography system. It provides real-time data on the UV absorption of the sample, which can be used to identify the presence and concentration of specific compounds. The Econo UV Monitor is a reliable and versatile tool for various applications in analytical and preparative chromatography.
Automatically generated - may contain errors
Lab products found in correlation
Sw41 rotor, by Beckman Coulter (10 mentions)
Gradient station, by BioComp Instruments (8 mentions)
Fc203b, by Gilson (8 mentions)
Sw28 rotor, by Beckman Coulter (7 mentions)
Piston gradient fractionator, by BioComp Instruments (6 mentions)
Superasin, by Thermo Fisher Scientific (6 mentions)
Piston gradient fractionator, by Bio-Rad (4 mentions)
Protease inhibitor, by Roche (3 mentions)
Protease inhibitor, by Thermo Fisher Scientific (3 mentions)
Sw41ti rotor, by Beckman Coulter (3 mentions)
Cycloheximide (chx), by Merck Group (3 mentions)
Flag tag (flag), by Merck Group (2 mentions)
A5892, by Merck Group (2 mentions)
Eif3b sc 16377, by Santa Cruz Biotechnology (2 mentions)
A 21277, by Thermo Fisher Scientific (2 mentions)
Econo gradient pump, by Bio-Rad (2 mentions)
Igor pro, by Wavemetrics (2 mentions)
Fc 203b fraction collector, by Gilson (2 mentions)
Complete edta free protease inhibitor cocktail, by Roche (2 mentions)
Fastprep 24 bead beater, by Biospec (2 mentions)
50 protocols using econo uv monitor
1
Polysome Profiling of Cellular Lysates
2
Polysome Extraction and Analysis from Plant Roots
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Polysomes were extracted from root tissue (0.3 g), which was flash frozen, ground in a mortar and pestle with liquid N2, and thawed in polysome extraction buffer (Chotewutmontri, Stiffler, Watkins, & Barkan, 2018 ) with modifications (50 mM Tris‐acetate (pH 8), 0.2 M KCl, 15 mM MgCl2, 0.2 M sucrose, 2 µg/ml pepstatin, 1 tablet/10 ml protease inhibitor, 2% polyoxyethylene‐10‐tridecyl ether, 1% Triton X‐100, 20 mM β‐mercaptoethanol, 3 mM DTT, 0.5 mg/ml heparin, 100 µg/ml chloramphenicol, 100 µg/ml cycloheximide). The homogenate was passed through a 40 µ filter followed by centrifugation at 4,700 g for 1 min at 4°C. The supernatant was collected and centrifuged at 21k g for 5 min at 4°C and centrifugation was repeated twice. The polysome extract was either used for fractionation immediately or flash frozen and stored at −80°C. The extract was layered onto 25%–65% sucrose gradient (50 mM Tris acetate (pH 8), 50 mM KCl, 15 mM MgCl2, 100 μg/ml cycloheximide, 100 μg/mL chloramphenicol) and centrifuged in a SW41 rotor (Beckman Coulter) at 35k g, 4°C, for 9 hr. Polysomes were fractionated using a Piston Gradient Fractionator (BioComp) equipped with a Econo UV monitor (Bio‐Rad) according to the manufacturer's instructions. Data were acquired using WinDAQ software (DATAQ Instruments).
3
Ribosome Profiling of YTHDC2 Knockdown
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We followed the reported protocols with the following modifications (31 (link), 32 (link)). CNE2-IRR cells were transduced with shCON, shYTHDC2_1#, and shYTHDC2_3# lentivirus and selected with puromycin (1 μg/ml). Before collection, cycloheximide (CHX) was added to the culture media at 100 mg/mL for 5 min. Fifty million cells from each group were harvested, rinsed in cold PBS with 100 mg/mL CHX, and quickly frozen in dry ice before lysis. The lysis buffer was formulated as 20 mM HEPES (pH7.6), One hundred mM KCl, 5 mM MgCl2, 100 mg/ml CHX, 1% Triton X-100, with freshly added 1:100 protease inhibitor (Roche) and 40 U/ml SUPERasin (Ambion). The sample was then fractionated into 24 fractions (0.5 mL per fraction) and analyzed by Gradient Station (BioCamp) equipped with ECONOUV monitor (BioRad, Hercules, CA) and Gilson FC203B fraction collector (Mandel Scientific, Guelph, Canada). RNA was purified from whole cell, 40S, 60S, 80S, and Polysome fractions and subjected to qPCR analysis. Expression of IGF1R in each fraction was normalized to TUBULIN as well as Input.
4
Polysome Profiling of Breast Cancer Cells
5
Polysome Analysis and Western Blotting
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Polysome analysis was performed as described previously [45 (link)]. After ultra-centrifugation, the mixture was fractionated and absorbance at 260 nm was recorded using a BioComp Piston Gradient Fractionator equipped with a Bio-Rad Econo UV Monitor. The corresponding fractions were further subjected to western blotting.
6
Polysome Profiling of HeLa Cells
7
Ribosome Fractionation by Sucrose Gradient
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The fraction of ribosome was separated by centrifugation in a sucrose gradient. Cells pretreated with 100 μg/ml cycloheximide were lysed in 1 ml lysis buffer (10 mM Tris, pH 7.4, 150 mM KCl, 5 mM MgCl2, 100 μg ml–1 CHX, 0.5% Triton-X-100, freshly add 1:100 protease inhibitor, 40 U ml–1 SUPERasin). After centrifugation at 15,000 g for 15 min, the supernatant was separated by 5/50% w/v sucrose gradient at 4 °C for 4 h at 140, 000 g (Beckman, rotor SW28). The sample was then fractioned and analyzed by Gradient Station (BioCamp) equipped with ECONO UV monitor (BioRad) and fraction collector (FC203B, Gilson). The fractions resulting from sucrose gradient were used for RNA extraction and qRT-PCR.
8
Profiling Ribosomes Bound to IGF2BP Proteins
9
Ribosomal subunit profiling in bacteria
10
Ribosome Profiling of Bacillus subtilis
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Early exponential phase cultures of B. subtilis strains grown in LB medium were treated with heat shock at 48 °C or 48 °C/53 °C for 15 min each. Samples of 50 mL were supplemented with 50 μg mL-1 chloramphenicol to stall translation and harvested by centrifugation at 4,000 x g for 10 min at 4 °C. Cells were resuspended in 25 mM HEPES-KOH, pH 7.5, 150 mM KOAc, 25 mM Mg(OAc)2, 1 mM dithiothreitol (DTT), n-Decyl−β−D-thiomaltopyranoside (DTM), 5% (w/v) sucrose) and lysed by sonication. The lysate was cleared by centrifugation at 16,000 x g for 15 min at 4 °C. 10 OD260 units were loaded on a 10 mL 5–45% (w/v) sucrose gradient prepared in the same buffer, run in a SW-40 Ti rotor (Beckman Coulter) at 57,471 x g for 16.5 h and analyzed using a Gradient Station (Biocomp) with an Econo UV Monitor (Bio-Rad).
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