BGC-823 cells were transfected with biotinylated miRNA (200 nM), harvested 24 h after transfection. Pull-down assay was performed as formerly described51 (link). Briefly, the cells were washed with PBS followed by brief vortex, and incubated in a lysis buffer on ice for 10 min. The lysates were precleared by centrifugation, and 50 μl of the samples were aliquoted for input. The remaining lysates were incubated with streptavidin magnetic beads (Thermo Scientific). After coated with RNase-free bovine serum albumin and yeast tRNA (both from Sigma), the beads were incubated at 4 °C for 3 h, washed twice with ice-cold lysis buffer, three times with the low salt buffer and once with the high salt buffer. The bound RNAs were purified using TRIzol and then for GCRL1 expression analysis by qRT-PCR.
Rnase free bovine serum albumin and yeast trna
Manufactured by Merck Group
Sourced in United States
RNase-free bovine serum albumin and yeast tRNA are laboratory reagents commonly used in molecular biology and biochemistry experiments. Bovine serum albumin (BSA) is a protein derived from bovine blood that is widely used as a stabilizer and blocking agent in various assays and procedures. Yeast tRNA is a type of transfer RNA isolated from yeast cells and is often used as a carrier or background nucleic acid in various applications. Both RNase-free BSA and yeast tRNA are produced under controlled conditions to ensure minimal RNase contamination, making them suitable for use in experiments involving RNA analysis and manipulation.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin magnetic beads, by Thermo Fisher Scientific (1 mentions)
Rna pull down lysis buffer, by Thermo Fisher Scientific (1 mentions)
Specific lysis buffer, by Thermo Fisher Scientific (1 mentions)
M 280 streptavidin magnetic beads, by Merck Group (1 mentions)
Rnase free dnase 1, by Roche (1 mentions)
Streptavidin agarose bead, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Dynabeads m 280 streptavidin beads, by Thermo Fisher Scientific (1 mentions)
Rnasin plus ribonuclease inhibitor, by Promega (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
6 protocols using rnase free bovine serum albumin and yeast trna
1
Biotin-Mimic Pull-Down of miRNA Targets
2
RNA Pulldown Identifies PCAT1-DKC1 Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA pull-down assay was used to identify the PCAT1 interaction with DKC1.
Briefly, before harvest, cells were transfected with 50 nM biotinylated RNA
probe for 48 h. Then the cells were washed with PBS and incubated for 10 min in
an RNA pull-down lysis buffer (Ambion, Austin, Texas, USA) on ice. The lysates
were precleared by centrifugation, and the samples (20 μl) were aliquoted for
input. The remaining lysates were incubated with M-280 streptavidin magnetic
beads pre-coated with RNase-free bovine serum albumin and yeast tRNA (Sigma, St.
Louis, MO, USA) at 4 °C for 3 h. After that, the beads were washed 2 times with
ice-cold lysis buffer and 3 times with an SDS-Tris low salt buffer (pH 8.0
containing 150 mM sodium chloride [NaCl]), and once with a high salt buffer
containing 500 mM NaCl. The bound complexes were purified for the following
analysis.
Briefly, before harvest, cells were transfected with 50 nM biotinylated RNA
probe for 48 h. Then the cells were washed with PBS and incubated for 10 min in
an RNA pull-down lysis buffer (Ambion, Austin, Texas, USA) on ice. The lysates
were precleared by centrifugation, and the samples (20 μl) were aliquoted for
input. The remaining lysates were incubated with M-280 streptavidin magnetic
beads pre-coated with RNase-free bovine serum albumin and yeast tRNA (Sigma, St.
Louis, MO, USA) at 4 °C for 3 h. After that, the beads were washed 2 times with
ice-cold lysis buffer and 3 times with an SDS-Tris low salt buffer (pH 8.0
containing 150 mM sodium chloride [NaCl]), and once with a high salt buffer
containing 500 mM NaCl. The bound complexes were purified for the following
analysis.
3
Biotin-labeled miR-1290 Pulldown Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NPC cells were transduced with 20 nM biotinylated wild type (WT)-bio-miR-1290 and MUT-bio-miR-1290. Forty-eight hours later, the cells were harvested and washed. According to the instructions of the kit, 20 mL streptavidin-coated magnetic beads (Dynabeads M-280, 11206D, Life Technology, Carlsbad, CA, USA) were activated. The magnetic beads were sealed with 10 mg/mL RNase-free bovine serum albumin and yeast tRNA (R8508, Sigma-Aldrich Chemical Company, St Louis, MO, USA) at 4°C for 30 min. The cells were then incubated for 10 min with a specific lysis buffer (Ambion, Austin, TX, USA). Next, the lysates experienced an incubation with M-280 streptavidin magnetic beads (Sigma, St Louis, MO, USA) for 2 h for RNA extraction. The bound RNA was purified by Trizol and the lncRNA ZNF667-AS1 enrichment was determined by RT-qPCR.
4
Investigating AFAP1-AS1 and miR-27b-3p Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The exact relationship between AFAP1-AS1 and miR-27b-3p was investigated by RNA pull-down assay, as described previously.[45 (link)] At first, that biotin-labeled AFAP1-AS1 wild-type (Bio-AFAP1-AS1-WT), biotin-labeled AFAP1-AS1-mutant RNA fragments (Bio-AFAP1-AS1-mut), and biotinylated negative control for biotin-labeled AFAP1-AS1 (NC-1), biotin-labeled miR-27b-3p wild-type (Bio-miR-27b-3p-WT), biotin-labeled miR-27b-3p-mutant (Bio-miR-27b-3p-Mut), and biotinylated negative control for biotin-labeled miR-27b-3p (NC-2) were respectively transfected into Hela for 48 h using Lipofectamine 2000 according to manufacturer procedures. Cells were lysed with RNase-free DNase I (Roche Applied Science, IN, USA) and complete protease inhibitor (Roche). Cell lysates were incubated with streptavidin agarose beads (Invitrogen) coated with RNase-free bovine serum albumin and yeast tRNA (both from Sigma-Aldrich). After an ice bath for 2 h, cell lysates were isolated (10,000 ×g for 10 min), and a pull-down assay was performed as described previously.[46 (link)] Levels of miR-27b-3p in the pull-down of wt-Bio-AFAP1-AS1, mut-Bio- AFAP1-AS1, and NC-1, as well as levels of AFAP1-AS1 in the pull-down of wt-Bio-27b-3p, mut-Bio-27b-3p, and NC-2, were quantified by real-time PCR.
5
Identifying miR-17 Targets in Melanoma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Melanoma stem cells (MDA-MB-435) were transfected with 50 nM of the synthesized biotin-miR-17 or biotin-miR-17-scrambled. At 36 h after transfection, the cells were incubated in lysis buffer (25 mM Tris-HCl, 100 mM KCl, 5 mM MgCl2, 0.3% NP-40, 50 U of RNasin Plus Ribonuclease Inhibitor [Promega], 1× Halt protease inhibitor cocktail [Thermo Fisher Scientific, USA]) for 20 min on ice. Dynabeads M-280 Streptavidin beads (Thermo Fisher Scientific) were activated according to the manufacturer’s protocol and blocked for 2 h at 4°C in lysis buffer containing 10 mg/mL RNase-free bovine serum albumin and yeast tRNA (Sigma-Aldrich, USA). After washing with lysis buffer, the beads were incubated with the lysate for 4 h at 4°C, followed by RNA extraction. The extracted RNAs were subjected to quantitative real-time PCR using miR-17-specific primers.
6
Profiling GAS5 Interactome by RNA and DNA Pull-Down
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The probes for GAS5 and its antisense RNA for RNA pull-down and DNA pull-down were designed and synthesized by Gzscbio Co. Ltd. (Guangzhou, China). Isolated HASMCs were washed in PBS, lysed in 0.5 mL of co-immunoprecipitation buffer, and incubated with 3 µg of biotinylated DNA oligo probes against a GAS5 back splice sequence at room temperature for 4 hours. Then, the HASMCs were incubated with streptavidin-coated magnetic beads (Invitrogen, SA10004) for another hour at room temperature. RNase-free bovine serum albumin and yeast tRNA (Sigma, Shanghai, China) were utilized to prevent the nonspecific binding of RNA and protein complexes. RNA complexes bound to beads were extracted by TRIzol for qRT-PCR analysis, and proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and silver stained. The specific bands were excised and analyzed by mass spectrometry.
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?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!