To perform the dsDNA reporter cleavage assays, 100 nM TmuRE-Ago complex and 100 nM guide RNA were incubated in a reaction buffer containing 20 mM HEPES (pH 7.5), 100 mM KAc, 5 mM Mg(Ac)2, and 2 mM DTT for 15 min at 37°C. Then, the indicated amount of target DNA and 500 nM dsDNA reporter were added to the reaction mixtures, unless otherwise specified. The reactions (10 μl) were incubated for 60 min and then terminated by adding 1 μl of 100 mM EDTA. The fluorescence signals were visualized and captured using a fluorescence imaging system (CLINX, China). Next, the reactions were transferred to a 384-well microplate, and the fluorescence was measured using a FlexStation 3 Multi-Mode Microplate Reader (excitation: 485 nm, emission: 525 nm).
Fluorescence imaging system
Manufactured by Clinx
Sourced in China
The Fluorescence imaging system is a laboratory equipment designed to capture and analyze fluorescent signals. It functions by illuminating samples with specific wavelengths of light, causing fluorescent molecules within the sample to emit light at a different wavelength, which is then detected and recorded by the system.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Protein ladder, by Thermo Fisher Scientific (1 mentions)
Ab7090, by Abcam (1 mentions)
Tbst solution, by Solarbio (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg h l, by Beyotime (1 mentions)
3 protocols using fluorescence imaging system
1
dsDNA Reporter Cleavage Assay
2
Nasal Mucosa Protein Extraction and Analysis
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Nasal mucosa tissues were cut and centrifuged to extract proteins using TRIzol reagent (Invitrogen, Carlsbad, California, U.S.A.). BCA kit (Thermo Scientific, Waltham, Massachusetts, U.S.A.) was used to measure protein concentration, and protein ladder (Invitrogen, Carlsbad, California, U.S.A.) was separated from protein by SDS/PAGE. Next, the proteins were transferred to PVDF membranes (Sigma–Aldrich, St. Louis, Missouri, U.S.A.), which were blocked by 5% albumin. The protein membranes were incubated first with primary antibody (Table 1 ) was used to incubate at 4°C for 12 h and then with secondary antibody (ab7090, Abcam, Cambridge, Massachusetts, U.S.A.) at 25°C for 3 h. Primary antibody and secondary antibody were dissolved in TBST solution (Solarbio, Beijing, China) following the instructions. ECL kit (Sigma–Aldrich, St. Louis, Missouri, U.S.A.) was used to stain the membranes in the dark. The protein flaser were taken by fluorescence imaging system (CliNX, Shanghai, China).
3
Western Blot Analysis of RAC2 Protein Expression
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The cell samples treated with the various compounds as described above were collected, subjected to protein determination by the Bradford method, and boiled at 100 °C for 5 min in loading buffer. The samples were then loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel at equivalent volumes for electrophoresis, and the proteins on the gel were transferred onto cellulose acetate membranes using transfer buffer solution. The samples were blocked with 5% skimmed milk powder for 1 h, and rabbit derived anti rat RAC2 antibody (Abcam, Cambridge, England) was added at 1:1000. The samples were placed in a refrigerator at 4 °C overnight, and the goat anti rabbit IgG (H+L) (Beyotime, ShangHai, China) labeled with horseradish peroxidase was added at 1:1000. After washing in Tris-buffered saline with Tween, the samples were subjected to color development by enhanced chemiluminescence and images were captured and analysis using Fluorescence imaging system (CLiNX, Shanghai, China). In addition, Image J (version 1.80, https://imagej.nih.gov/i j/) was used to measure and analyze the gray level of WB expression.
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