All processes were performed in the RNase-free conditions. For antisense oligomer affinity pull-down assay, sense or antisense biotin-labeled DNA oligomers corresponding to LAST or CCND1 mRNA (1 μM) were incubated with lysates from HCT116 cells (1 × 107) or the cytosolic/nuclear extracts. One hour after incubation, streptavidin-coupled agarose beads (Invitrogen) were added to isolate the RNA-protein complex or RNA-RNA complex. For in vitro RNA pull-down assay, 5 μg in vitro-synthesized biotin-labeled RNA was incubated with lysates from HCT116 cells (1 × 107) for 3 hr. streptavidin-coupled agarose beads (Invitrogen) were then added to the reaction mix to isolate the RNA-protein complex or RNA-RNA complex. Immunocomplexes were then analyzed by real-time RT-PCR or western blotting.
Streptavidin coupled agarose beads
Manufactured by Thermo Fisher Scientific
Sourced in China, United States
Streptavidin-coupled agarose beads are a type of affinity chromatography resin. The beads are made of agarose and have streptavidin, a protein that binds to biotin, covalently attached to the surface. These beads can be used to capture and purify biotinylated molecules from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Cell extraction buffer, by Thermo Fisher Scientific (4 mentions)
T7 rna polymerase, by Roche (3 mentions)
Biotin rna labeling mix, by Roche (3 mentions)
Biotin, by Roche (2 mentions)
Rnasin, by Promega (2 mentions)
Biotin, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Scramble control, by RiboBio (1 mentions)
Biotin, by RiboBio (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Piercetm magnetic rna protein pull down kit, by Thermo Fisher Scientific (1 mentions)
Anti igg, by Santa Cruz Biotechnology (1 mentions)
Protein a g agarose, by Santa Cruz Biotechnology (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rnase free dnase 1, by Roche (1 mentions)
27 protocols using streptavidin coupled agarose beads
1
Biotin-based RNA-protein/RNA complex isolation
2
Lnc-PDZD7 RNA-Protein Interactions
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All processes were performed in RNase-free conditions. For the antisense oligomer affinity pull-down assay, sense or antisense biotin-labeled DNA oligomers corresponding to Lnc-PDZD7 (1 mM) were incubated with lysates from HCC cells. One hour after incubation, streptavidin-coupled agarose beads (Invitrogen, Shanghai, China) were added to isolate the RNA-protein complex or RNA-RNA complex. For the in vitro RNA pull-down assay, 5 mg in vitro-synthesized biotin-labeled DNA was incubated with lysates for 3 h. streptavidin-coupled agarose beads (Invitrogen, Shanghai, China) were then added to the reaction mix to isolate the RNA-protein complex or RNA-RNA complex. Immunocomplexes were then analyzed by real-time RT-PCR or Western blotting.
3
Biotin-Streptavidin Pulldown for YB-1 Promoter Binding
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To screen for proteins bound to the YB-1 promoter, a biotin-streptavidin pulldown assay was conducted. The YB-1 promoter was labeled with biotin (Thermo Scientific, USA). MCF-7 breast CSCs were harvested and washed with cold PBS three times. Then, cells were lysed in lysis buffer (5 mM piperazine-1,4-bisethanesulfonic acid, 85 mM KCl, and 0.5% Nonidet P-40) containing 2 mM phenylmethanesulfonyl fluoride (PMSF) and were then incubated for 30 min on ice. The cell lysate was incubated with the biotin-labeled YB-1 promoter and streptavidin-coupled agarose beads (Thermo Scientific, USA) at 4 °C overnight. After three washes with PBS, the mixture was resuspended in RIPA lysis buffer (Beyotime Biotechnology, China) and heated at 100 °C for 5 min. The mixture was centrifuged for 5 min at 15,000×g, and the supernatant was analyzed by western blotting.
4
BTK Occupancy Assay in Ramos Cells
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Example 281
BTK Occupancy in Ramos Cells with I-7 and I-215 Using Streptavidin Beads
Ramos cells were incubated with 0.1, 0.05, 0.01, or 0.001 μM I-7 in serum free media for 1 hour at 37° C. The cells were pelleted by centrifugation and lysed in Cell Extraction buffer (Invitrogen) for 10 minutes on ice, centrifuged (10 minutes at 14,000 rpm) and the supernatant was collected. Cell lysates were incubated with 1 μM I-215 for 1 hour at room temperature, then incubated with streptavidin-coupled agarose beads (ThermoFisher) overnight at 4° C. The beads were washed three times with lysis buffer and the bound proteins were boiled off the beads at 95° C. for 5 minutes in 4×LDS Sample Buffer. The amount of BTK associated with the probe I-215 was assessed by BTK western blot. All values were normalized to the DMSO-treated sample which is set to 100%.
5
BTK Occupancy Assay in Ramos Cells
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Example 296
BTK Occupancy in Ramos Cells With I-7 and I-215 Using Streptavidin Beads
Ramos cells were incubated with 0.1, 0.05, 0.01, or 0.001 μM I-7 in serum free media for 1 hour at 37° C. The cells were pelleted by centrifugation and lysed in Cell Extraction buffer (Invitrogen) for 10 minutes on ice, centrifuged (10 minutes at 14,000 rpm) and the supernatant was collected. Cell lysates were incubated with 1 μM I-215 for 1 hour at room temperature, then incubated with streptavidin-coupled agarose beads (ThermoFisher) overnight at 4° C. The beads were washed three times with lysis buffer and the bound proteins were boiled off the beads at 95° C. for 5 minutes in 4×LDS Sample Buffer. The amount of BTK associated with the probe I-215 was assessed by BTK western blot. All values were normalized to the DMSO-treated sample which is set to 100%.
6
Biotin-miR-31-5p pulldown assay
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For RNA pull-down assay, Biotin-labeled miR-miR-31-5p (Sangon, Shanghai, China) oligo was first transfected into 786-0 and A498 cells. Following 2-day cultivation, 786-0 and A498 cells were lysed and then incubated with streptavidin-coupled agarose beads (Thermo Fisher Scientific) to pull down the complex. The amount of TUG1 was detected by qRT-PCR.
7
NEAT1 RNA Pull-down Assay for miR-338-3p
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For the RNA pull-down assay, FLSs lysates were incubated with biotin-labeled Scramble control, Scramble control, sense, antisense NEAT1 probe synthesized by RiboBio Co. Ltd (Guangzhou, China). The mixture was incubated for 2 h. After incubation of this mixture for 2 h, streptavidin-coupled agarose beads (ThermoFisher, Shanghai, China) were added for 2 h. After washing with PBS, the miR-338-3p from the pulled-down RNA-RNA complexes was measured by qRT-PCR. Experiments were performed in triplicate and repeated three times.
8
miR-451a Pulldown and qPCR Analysis
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Biotin-labeled miR-451a probe and biotin-labled NC probe were transfected into cells and incubated for 48 hours. And then, cells were incubated in lysis buffer with streptavidin-coupled agarose beads (Thermo Fisher) to pull down the complex. After the complex was washed, RNAs were isolated and measured using RT-qPCR. The experiment was conducted at least 3 times.
9
RNA Pull-Down Assay for miR-34a-5p
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RNA pull‐down assay was performed according to previous description.18 Scramble control, TUG1 sense, and antisense probes were biotin‐labeled from RiboBio Co. Ltd (Guangzhou, China). FLS cell lysates were prepared by RIPA buffer followed by incubation with probes for 2 h. Streptavidin‐coupled agarose beads (Thermo Fisher Scientific, Shanghai, China) were added into the reactions for 2 h to pull down the RNA‐RNA complex. The enrichment of miR‐34a‐5p in the RNA‐RNA complex was determined by qRT‐PCR. Experiments were performed in triplicate and repeated three times.
10
BTK Occupancy Assay with I-7 and I-215
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Example 296
BTK Occupancy in Ramos Cells with I-7 and I-215 Using Streptavidin Beads
Ramos cells were incubated with 0.1, 0.05, 0.01, or 0.001 μM I-7 in serum free media for 1 hour at 37° C. The cells were pelleted by centrifugation and lysed in Cell Extraction buffer (Invitrogen) for 10 minutes on ice, centrifuged (10 minutes at 14,000 rpm) and the supernatant was collected. Cell lysates were incubated with 1 μM I-215 for 1 hour at room temperature, then incubated with streptavidin-coupled agarose beads (ThermoFisher) overnight at 4° C. The beads were washed three times with lysis buffer and the bound proteins were boiled off the beads at 95° C. for 5 minutes in 4×LDS Sample Buffer. The amount of BTK associated with the probe I-215 was assessed by BTK western blot. All values were normalized to the DMSO-treated sample which is set to 100%.
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