6xHisSBP‐tagged SidP, MavQ, and Lem14 were purified using the 6xHis‐tag, as described above, followed by an additional SBP‐tag purification step with streptavidin mag sepharose (GE Healthcare Life Sciences) and eluted in 50 mM HEPES pH 7.5, 300 mM NaCl, 2.5% glycerol, and 4 mM biotin. The lipid overlay blot was performed as described (Weber et al, 2013 ), with minor modifications. Briefly, the PIP strips (Echelon, P‐6001) were blocked in 3% fatty acid‐free BSA (Roche) in Tris‐buffered saline with 0.1% Tween 20 (blocking buffer) and incubated overnight in the dark at 4°C with 5 pmol/ml purified protein in a total volume of 10 ml blocking buffer. The PIP strips were washed with blocking buffer, incubated with mouse anti‐polyhis antibody (1:2,000, clone HIS1, Sigma‐Aldrich) in blocking buffer, washed, and incubated with anti‐mouse HRP antibody (7076, Cell Signaling Technologies) in blocking buffer.
Streptavidin mag sepharose
Manufactured by GE Healthcare
Sourced in United States
Streptavidin Mag Sepharose is a magnetic bead-based separation media. It utilizes the high-affinity interaction between streptavidin and biotin to capture and isolate biotinylated molecules. The magnetic properties allow for efficient separation of the target from the sample matrix.
Automatically generated - may contain errors
Lab products found in correlation
Ampliscribe t7 flash biotin rna transcription kit, by Illumina (3 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Pip strip, by Echelon Biosciences (1 mentions)
Fatty acid free bsa, by Roche (1 mentions)
Pip strips, by Merck Group (1 mentions)
Bioruptor sonicator, by Diagenode (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Superscript 3 supermix, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Qiagen (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Goat anti rabbit igg hrp antibody, by Santa Cruz Biotechnology (1 mentions)
Rat monoclonal anti ha antibody clone 3f10, by Roche (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Dynabeads, by STEMCELL (1 mentions)
Anti actin antibody, by Santa Cruz Biotechnology (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Merck Group (1 mentions)
Horseradish peroxidase linked anti goat igg, by Santa Cruz Biotechnology (1 mentions)
Azide peg3 biotin conjugate, by Merck Group (1 mentions)
Dopac, by Merck Group (1 mentions)
Benzyl azide, by Fujifilm (1 mentions)
15 protocols using streptavidin mag sepharose
1
Purification and Lipid Overlay Assay
2
Ikaros-Bound Chromatin Profiling
3
Affinity Purification of Biotinylated Peptides
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
500μg of each peptide, in lysis buffer, was bound to streptavidin-conjugated magnetic sepharose beads (Streptavidin-MagSepharose, GE Healthcare) by incubation at 4°C on a rotating wheel for 1h, then washed three times with lysis buffer.
The cell extract was pre-cleared by incubation with empty streptavidin conjugated magnetic beads at 4°C on a rotating wheel for 1h.
After removal of the pre-clearing beads, the extract was incubated at 4°C on a rotating wheel for a further 2h with each of the biotinylated peptides bound to streptavidin-conjugated magnetic sepharose beads.
The beads were washed three times with lysis buffer without protease inhibitors, transferred to fresh eppendorf tubes and washed twice more with lysis buffer without either protease inhibitors or Triton-x-100.
5% of the beads were taken for western blot analysis and the remainder were subjected to trypsin-digest and the products analysed by mass spectroscopy, as described previously [45 (link)].
The cell extract was pre-cleared by incubation with empty streptavidin conjugated magnetic beads at 4°C on a rotating wheel for 1h.
After removal of the pre-clearing beads, the extract was incubated at 4°C on a rotating wheel for a further 2h with each of the biotinylated peptides bound to streptavidin-conjugated magnetic sepharose beads.
The beads were washed three times with lysis buffer without protease inhibitors, transferred to fresh eppendorf tubes and washed twice more with lysis buffer without either protease inhibitors or Triton-x-100.
5% of the beads were taken for western blot analysis and the remainder were subjected to trypsin-digest and the products analysed by mass spectroscopy, as described previously [45 (link)].
4
RNA-Protein Interaction Profiling Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Biotin-labeled RNAs were transcribed with AmpliScribe T7-Flash Biotin-RNA Tran-scription Kit (Epicentre) and incubated with RNase-free DNase I and purified with RNeasy Mini Kit (Qiagen). The lambda transcript was generated with the control plasmid provided by the Transcription Kit. Biotinylated RNA supplied with RNA structure buffer (10 mM Tris pH 7, 0.1 M KCl and10 mM MgCl2) was heated to 90 °C for 2 min, incubated on ice for 2 min and then shifted to room temperature (RT) for 20 min. Biotinylated RNAs were mixed with purified proteins and incubated for 1 h. Then, the RNAs were incubated by Streptavidin Mag Sepharose (GE Healthcare) for 1 h. After washing, the pull-down assay was measured by PCR.
5
Biotinylated RNA Pull-Down Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The biotinylated RNA pull-down assay was performed as described previously68 (link)69 (link). Briefly, biotin-labelled RNAs was in vitro transcribed with AmpliScribe T7-Flash Biotin-RNA Transcription Kit (Epicentre), treated with RNase-free DNase I and purified with an RNeasy Mini Kit (Qiagen). The lambda transcript was generated with the control plasmid provided by the Transcription Kit. To form the proper secondary structure, biotinylated RNA supplied with RNA structure buffer (10 mM Tris pH 7, 0.1 M KCl and 10 mM MgCl2) was heated to 90 °C for 2 min, incubated on ice for 2 min and then shifted to room temperature (RT) for 20 min. The RNA was then mixed with hypoxic HeLa nuclear extract or purified proteins and incubated at RT for 1 h, followed by incubating with Streptavidin Mag Sepharose (GE Healthcare) at RT for 1 h. After subsequent washes, the pull-down complexes were analysed by standard western blot technique.
6
Immobilization of Purified VC1-His-Strep
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified VC1-His-Strep was immobilized on streptavidin-coated magnetic beads by exploiting the affinity of the Strep tag for Streptavidin (Streptavidin Mag Sepharose™, GE Healthcare, cat. N. 28-9857-99). The binding capacity of the beads was 3 μg of VC1-His-Strep/10 μl of medium slurry. In general, 50 μg of purified VC1-His-Strep in 170 μl of buffer C were added to 5 μl of packed streptavidin coated-beads previously obtained by washing 50 μl of 10% slurry with Buffer C, and incubated for 1 h at 4 °C on a rotary mixer at low speed. The unbound material was carefully removed and the magnetic beads were washed with 500 μl of buffer C and immediately used or stored at 4 °C. For simplicity, the VC1 bound to the beads is referred to as the VC1-resin and the beads alone as the control resin.
7
Biotin-Conjugated E-Box Oligonucleotide Pull-Down for VE-Cadherin Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Based on ChIP assay, we synthesized a pair of biotin-conjugated oligonucleotides containing the E-box site of VE-cadherin gene promoter. The sequences were: forward, 5′-GAC-TGG-GCT-CAC-CCC-AGA-TCA-GCT-GAT-TTG-GAA-TCT-CCC-3′; reverse, 5′- GGG-AGA-TTC-CAA-ATC-AGC-TGA-TCT-GGG-GTG-AGC-CCA-GTC-3′. The oligonucleotides were incubated 16 h at 4°C with 300 μg of nuclear extracts isolated from PBS or OPN-treated HUVECs. The reaction mixtures were then added with 20 μl of Streptavidin Mag Sepharose (GE Healthcare, Piscataway, NJ, USA) and incubated on a rotatory shaker for another 3 h. E-box-associated proteins were pulled down after centrifugation at 3000×g for 5 min at 4°C and subjected to immunoblot analyses.
8
Comprehensive Chromatin Interactome Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIRP was performed by following the previous protocol (29 (link)). In the beginning, we designed anti-sense oligo probes of PTTG3P and LacZ with biotin-labeled at 3′-prime end by ChIRP Probe Designer. Forty million H1299 cells were treated with 65 ng/ml nocodazole for G2/M synchronization followed by 1% glutaraldehyde for 10 minutes at room temperature for crosslinking. After lysing crosslinking, DNA was sheared to 200–500 bp at 4°C and hybridized with biotinylated target probes in 37°C. Chromatin bound with probes were isolated by Streptavidin Mag Sepharose (GE Healthcare) and individually extracted DNA, RNA, and protein for further detection as previously described (29 (link)). Sequences of biotinylated probes used in ChIRP were listed in Supplementary Table S3
9
Protein-Aptamer Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Biotinylated aptamers (4 μM; 2c2s or negative control) were immobilized on Streptavidin Mag Sepharose (GE Healthcare, Chicago, IL, USA) by incubation in PBS for 20 min in RT. After washing with PBS, the beads were incubated with 0,2% BSA (BioShop Canada Inc., Burlington, VT, Canada) in the SELEX buffer for 30 min at RT to block unspecific sites and washed with SELEX buffer. Next, the beads were suspended in SELEX buffer containing 40 μg/mL tRNA and incubated with PD-L1 (18–239 or 18–134 Cterm Histag; final concentrations: 90, 45, and 22,2 μg/mL) for 20 min at RT while mixing continuously. After incubation, the beads were washed with SELEX buffer and the bound protein was eluted by boiling briefly in the loading buffer (3% SDS, 10% glycerol, 12,5 mM Tris-HCl, 100 mM DTT, 0,05% bromophenol blue, pH 6.8). The recovered proteins were analyzed by SDS/PAGE. Coomassie blue-stained gels were imaged using ChemiDoc (Bio-Rad Laboratories, Inc. Hercules, CA, USA).
10
Biotin-labeled DNA Pulldown Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Biotin labeled DNAs were synthesized by Integrated DNA Technologies (Coralville, IA, USA). 20 million HEK-293T cells were lysed and incubated with 10 µg biotin-labeled DNAs overnight. The RNAs associated with biotin-labeled DNAs were then pulled down with Streptavidin Mag Sepharose (GE Healthcare, Madison, WI, USA) after a 1-hour incubation. RNAs was then washed and purified by phenol chloroform extraction. 20 µl RNAse-free water was used to re-suspend the RNA and 6 µl RNA was used for cDNA synthesis with random primers using SuperScript III SuperMix (Invitrogen, Grand Island, NY, USA).
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!