A biotinylated RNA probe was synthesized using an in vitro transcription system (Promega) according to the manufacturer’s instructions. The cells were lysed using RIPA lysis buffer, and total cell lysates were pre-cleaned using streptavidin beads (Sigma-Aldrich) at 4 °C for 1 h. The lysates were then diluted with 5 × EMSA buffer (50 mM Hepes/pH 8.0, 250 mM KCl, 10 mM MgCl2, 5 mM DTT, 25% glycerol, 0.5 mg/ml tRNA, 0.7 mg/ml heparin, 100 unit/ml RNase inhibitor, and protease inhibitors) and incubated with biotin-labeled RNA probes at 4 °C for 2 h. The mixtures were subjected to UV-crosslinking (120 mJ/cm2) three times for 5 min each and then incubated with streptavidin beads while rotating at 4 °C for 1 h. The beads were washed three times with wash solution (10 mM Hepes/pH 8.0, 40 mM KCl, 3 mM MgCl2, 2 mM DTT, 5%, glycerol, 0.5% SDS and 2% NP-40) and then subjected to western blot analysis.
Streptavidin beads
Manufactured by Merck Group
Sourced in United States
Streptavidin beads are solid-phase supports coated with the protein streptavidin. Streptavidin has a high affinity for the small molecule biotin, allowing for the capture and purification of biotinylated molecules such as proteins, nucleic acids, and cells.
Automatically generated - may contain errors
Lab products found in correlation
In vitro transcription system, by Promega (2 mentions)
Biotin rna labeling mix, by Roche (2 mentions)
T7 rna polymerase, by Promega (2 mentions)
Streptavidin agarose beads, by Merck Group (2 mentions)
Auxin, by Merck Group (2 mentions)
Rnase free dnase 1, by Roche (1 mentions)
Rneasey mini kit, by Qiagen (1 mentions)
T7 rna polymerase, by Roche (1 mentions)
Pgem t vector, by Promega (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Amicon ultra spin column, by Merck Group (1 mentions)
Coomassie brilliant blue, by Merck Group (1 mentions)
Tev protease, by Thermo Fisher Scientific (1 mentions)
Tev protease, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Pyromark q24, by Qiagen (1 mentions)
3xflag peptide, by Merck Group (1 mentions)
Dnajb1, by Abcam (1 mentions)
Hsp90, by R&D Systems (1 mentions)
V5 peptide, by Merck Group (1 mentions)
58 protocols using streptavidin beads
1
RNA-Protein Interaction Profiling
2
Linc-KILH RNA-Protein Interaction Profiling
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RNA pull-down was performed as previously described 20 (link). Briefly, biotin-labeled Linc-KILH and control RNA were produced in vitro using Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Promega) and then were purified using an RNeasey Mini Kit (Qiagen, Valencia, CA, USA) after treatment with RNase-free DNase I (Roche). Three μg biotinylated RNAs were mixed with proteins extracted from Huh7 cells, followed by targeting RNAs with streptavidin beads (Millipore, Bedford, MA, USA). Finally, the retrieved proteins were washed with a RIPA buffer and subjected into SDS-PAGE for separation, followed by Western blotting.
3
Histone Peptide Binding Assay for RACK7
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For peptide pull-down assays, recombinant, full-length HA-tagged RACK7 and mutants were purified from insect cell Sf9, while PHD, Bromo, and PWWP domains of RACK7 were purified from E. coli. Two micrograms of full-length RACK7 or mutant proteins was incubated with 2 μl (concentration, 0.1 mM) of various biotinylated histone peptides in the binding buffer [20 mM tris-HCl (pH 7.3), 150 mM NaCl, 0.1% NP-40] at 4°C for 4 hours. The protein-peptide complexes were immobilized to streptavidin beads (Millipore) at 4°C for 1 hour. The bound proteins were washed with binding buffer and separated on 10% SDS–polyacrylamide gel electrophoresis (PAGE) followed by Coomassie blue staining. Modified histone peptides were synthesized by Beijing Scilight Biotechnology LLC.
4
Identification of Irm RNA-Binding Proteins
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RNA pull down was performed as previously described39 (link). In brief, the pGEM-T vectors (Promega, Madison, WI, USA) carrying Irm, antisense of Irm, truncated fragments of Irm or GAPDH DNAs were linearized with the corresponding restriction enzymes to prepare the template DNAs for in vitro transcription. Then, biotin-labeled RNA transcripts were transcribed in vitro using T7 RNA polymerase (Roche, Basle, Switzerland). Three micrograms of in vitro biotinylated RNAs were mixed with proteins extracted from C2C12 cells, followed by targeting the RNAs with streptavidin beads (Millipore, Bedford, MA). The coprecipitated proteins were then visualized by western blotting.
5
Streptavidin Bead Affinity Purification
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Streptavidin beads (Millipore, Cat# 16-126) were pre-washed with cell lysis buffer before incubation with biotinylated GAR-un/Rme2s peptides (10 µg) in 500 µl cell lysis buffer for 1 h at 4°C with rocking for conjugation. Lysis buffer was composed of the following Sigma compounds: 50 mM sodium diphosphate (Cat# S0751-100G), 300 mM sodium chloride (Cat# S9888-25G), 10 mM imidazole (Cat# I5513-25G) all adjusted to pH 8.0. The conjugated peptide-beads complex was then incubated with cell lysates prepared from primary MEFs for 1 h at 4°C with rocking. After incubation, the bound proteins were eluted by addition of SDS-Lammeli buffer for western blot analysis. GAR peptide sequences are as follows with SDMA marked by ‘*’:
GAR-un peptide: GGRGRGGGFRGRGRGGGG-BIOTIN
GAR-Rme2s peptide: GG[R**]G[R**]GGGF[R**]G[R**]G[R**]GGGG-BIOTIN
GAR-un peptide: GGRGRGGGFRGRGRGGGG-BIOTIN
GAR-Rme2s peptide: GG[R**]G[R**]GGGF[R**]G[R**]G[R**]GGGG-BIOTIN
6
Analyzing Replisome Dynamics with iPOND
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iPOND assays were performed as previously described17 (link). Briefly, cells were treated with the following conditions—10 μM EdU for 15 min, 10 μM EdU followed by 15 μM Thymidine for 1 hr and 10 μM EdU followed by 2 mM HU for 1 hr. Cells were subsequently fixed in 1% formaldehyde solution, quenched with glycine, permeabilized with 0.25% Triton X-100 and clicked with biotin azide. Cell pellets were lysed using 1% SDS in 50 mM Tris-HCl (pH 8) and pull down was performed for 2 hr in 4 °C using 50 μl/sample Streptavidin beads (millipore). Beads were subsequently washed once with 1 ml lysis buffer (5 min), 1 ml low-salt buffer (1% Triton X-100, 20 mM Tris [pH 8.0], 2 mM EDTA, 150 mM NaCl; 5 min), 1 ml high-salt buffer (1% Triton X-100, 20 mM Tris [pH 8.0], 2 mM EDTA, 500 mM NaCl; minutes) and finally twice with 1 ml lysis buffer (5 min). Washed beads were resuspended in 30 μl of 2X Laemmli buffer (BioRad), heated at 100 °C for 10 min and proceeded for immunoblotting.
7
Peptide Pull-Down Assay for BAG3 Interactions
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Peptide pull-down assays were performed as described previously [18 (link)]. Briefly, streptavidin agarose beads (Millipore, Burlington, MA, USA) were pre-washed once with 1× mild buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 0.1% NP-40, 5 mM EDTA, 5 mM EGTA, 15 mM MgCl2), and 15 μg of either biotinylated LFV-Z-WT or LFV-Z PPxY mutant peptide was incubated with the pre-washed streptavidin beads in 500 μL of 1× mild buffer for 1 h at 4 °C with rocking. The beads were washed 3X with mild buffer and then incubated with HEK293T cell extracts containing either BAG3-WT, BAG3-ΔN, or BAG3-ΔC. The beads were then washed 3X with 1× mild buffer and suspended in 30 μL of 2× loading buffer with boiling. BAG3 proteins were detected by Western blotting using anti-Myc antibody.
8
Purification of Human 26S Proteasomes
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Human 26S proteasomes were purified using a stable HEK293 cell line harboring biotin-tagged human β4 as previously described with slight modification.6 (link) The cells were cultured in 15-cm culture dishes, and Dounce-homogenized in lysis buffer (100 mM NaCl, 50 mM NaH2PO4 (pH 7.5), 10% glycerol, 5 mM MgCl2, 0.5% NP40, 5 mM ATP, 1 mM DTT) containing protease inhibitors. After centrifugation, the supernatants were incubated with streptavidin agarose bead (Millipore, Darmstadt, Germany) for 5 h at 4 °C. The beads washed with lysis buffer and TEV buffer (50 mM Tris-HCl (pH 7.5) containing 1 mM ATP and 10% glycerol). The 26S proteasome was eluted from the beads using TEV protease (Invitrogen, Waltham, MA, USA) in TEV buffer. To cleavage proteasome from streptavidin beads, using TEV protease containing 1 mM ATP for 1 h at 30 °C and was concentrated using an Amicon ultra-spin column (Millipore). The purified proteasomes were separated by a 4–20% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and routinely checked for their size and purity by the EzWay silver staining kit (Koma Biotech, Seoul, Korea) or Coomassie brilliant blue (CBB, Sigma-Aldrich, St Louis, MO, USA) staining.
9
Competitive Binding Assay for 53BP1 Tudor
10
Detecting Newly Synthesized Proteins in Autophagy
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HeLa cells were labeled with AHA to detect the newly synthesized proteins as previously described.12 AHA (50 µM) was used to lable cells in L‐methionine‐free DMEM (Invitrogen, 21013). To induce autophagy, cells were starved for 2 h. CHX (cycloheximide, 10 µM) was added to inhibit protein synthesis. The AHA‐incorporated proteins were enriched through a click reaction with TAMRA alkyne or alkyne‐bearing biotin. Affinity purification was further performed through adding 50 µL streptavidin beads (Sigma‐Aldrich, S1638). The enriched proteins were either visualized using gel electrophoresis or subjected to iTRAQ labeling for protein identification and quantification using LC‐MS/MS (liquid chromatography‐tandem MS).
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