We prepared 20 ul reaction mixtures, which contained 160 nM 5’ 6FAM-labeled RNA oligos of both rG4FSs and non-rG4FSs (Supplementary data S1, Table S3 ), and binding buffer consisting of 10 mM Tris–HCl pH 8.0, 150 mM KCl, 1 mM EDTA, 2 mM MgCl2, 10% glycerol, 2 mM DTT, 0.1 mg/ml Ultrapure-BSA (Invitrogen, AM2616) and Ribolock (1:40; Thermo Scientific, E00381), with or without recombinant human G3BP1 protein (Prospec, Enz-048). We incubated the binding reactions at 37°C for 1 h and then loaded them onto a 5% native non-denaturing polyacrylamide (acrylamide:bis-acrylamide 29:1 (30%); Bio-Lab, UN3426) gel consisting of (for 12.5 ml) 9 ml DEPC-ddW, 1.25 ml TBE ×10, 2.075 ml 30% polyacrylamide, 125 ul 10% ammonium persulfate (APS; Bio-Rad, 1610700) and 12.5 ul TEMED (Bio-Rad, 1610801). We performed gel electrophoresis at 100 V for 50 min in TBEx1 buffer on ice and in the dark. After 50 min, we performed gel scanning using ImageQuant LAS4000 (GE Healthcare) gel imager at cy2 channel (488 nm). For the EMSA experiments with the oligo r(AGG)5 we then electro-transferred the RNA-protein complex from the gel to a nitrocellulose membrane (Whatmann; 10401383) and used primary anti-G3BP1 antibody (Santa cruz; sc-365338) to detect the recombinant hG3BP1 within the lanes.
Ultrapure bsa
Manufactured by Thermo Fisher Scientific
Sourced in United States
UltraPure BSA is a high-quality bovine serum albumin product designed for use in various laboratory applications. It is a purified, lyophilized powder that provides a consistent and reliable source of albumin protein.
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Lab products found in correlation
Perfecthyb plus hybridization buffer, by Merck Group (3 mentions)
Immedge pen, by Vector Laboratories (2 mentions)
Chromium next gem single cell 5 beads, by 10x Genomics (2 mentions)
Superase in rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Ultrapure salmon sperm dna, by Thermo Fisher Scientific (2 mentions)
E coli trna, by Merck Group (2 mentions)
Dead cell removal kit, by Miltenyi Biotec (2 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Ribolock, by Thermo Fisher Scientific (1 mentions)
Temed, by Bio-Rad (1 mentions)
Superscript 3 rt platinum taq mix, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 2 system, by Roche (1 mentions)
Platinum taq polymerase, by Thermo Fisher Scientific (1 mentions)
Streptavidin sepharose bead, by GE Healthcare (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Salmon sperm dna, by Thermo Fisher Scientific (1 mentions)
Yeast trna, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Illumina (1 mentions)
Nupage tris acetate gel, by Thermo Fisher Scientific (1 mentions)
Riboguard, by Illumina (1 mentions)
37 protocols using ultrapure bsa
1
G3BP1 Binding to RNA G-Quadruplex Structures
2
Immunofluorescence Staining for Protein Localization
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Slides were washed in 1% Triton X-100 in RNase-free 1X PBS and rocked gently for 10 min. Slides were washed twice for 5 min in RNase-free 1X PBS the sample perimeter of each slide was marked with an ImmEdge pen (Vector Laboratories #H-4000) in between both washes. 250 µL of blocking solution (0.5% UltraPure BSA (Invitrogen #AM2616) in RNase-free 1X PBS) was added to the sample area of each slide and the slides were incubated at room temperature in a dark humid chamber for 1 h, shaking gently. Using coverslips, slides were incubated with 40 µL of diluted primary antibody in blocking solution overnight in a dark, sealed, humid chamber at 4°C. Slides were washed 3 times for 5 min in RNase-free 1X PBS. 40ul of diluted secondary antibody in blocking solution was applied to each slide (including coverslips) and slides were incubated for 2 h in a dark humid chamber at room temperature. Slides were washed 3 times for 5 min in RNase-free 1X PBS. 250 µL of post-fixative (4% paraformaldehyde in RNase-free 1X PBS) was added to each sample area for 3 min at room temperature. Each slide was then washed 3 times for 5 min in RNase-free 1X PBS.
3
One-Step RT-PCR for E and GUSB Genes
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The one-step reverse transcriptase reaction for the detection of the E gene was performed in a 25 μL reaction volume as described previously [2 ]. The assay was also performed in a 10 μL reaction volume, consisting of 5 μL 2× reaction buffer, 0.16 μL of a 50 mM magnesium sulfate solution and 0.40 μL of SuperScript™ III RT/Platinum™ Taq Mix (all provided by the SuperScript™ III One-Step RT-PCR with Platinum™ Taq Polymerase, Invitrogen, USA), 400 nM forward primer, 400 nM reverse primer, 200 nM probe (primers and probe provided by TIB MolBiol, Berlin, Germany), 0.4 μg of nonacetylated bovine serum albumin (Ultrapure™ BSA, Invitrogen, USA), and 2 μL of RNA.
The E-GUSB duplex RT-PCR was performed in a 25 μL and 10 μL reaction volume as described above, with the addition of 1.25 μL and 0.5 μL of the GUSB Gene Expression assay (Bio-Rad Laboratories, Hercules, CA), respectively.
All assays were performed on the LightCycler 480 II system (Roche Diagnostics) using the following cycling conditions: 10 min at 55 °C for reverse transcription, followed by 3 min at 95 °C, continuing with 15 s at 95 °C, and 30 s at 58 °C.
The E-GUSB duplex RT-PCR was performed in a 25 μL and 10 μL reaction volume as described above, with the addition of 1.25 μL and 0.5 μL of the GUSB Gene Expression assay (Bio-Rad Laboratories, Hercules, CA), respectively.
All assays were performed on the LightCycler 480 II system (Roche Diagnostics) using the following cycling conditions: 10 min at 55 °C for reverse transcription, followed by 3 min at 95 °C, continuing with 15 s at 95 °C, and 30 s at 58 °C.
4
RNA-Protein Interaction Assay Protocol
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A 30 µL of HeLa nuclear extract (purchased from Computer Cell Culture Centre, prepared according to the classical Dignam protocol) were diluted 10 times in binding buffer (20 mM Tris pH 8.0, 100 mM NaCl, 5 mM MgCl2, 0.4% NP40) and supplemented with protease inhibitors (Roche), 60 µg of yeast tRNA (Invitrogen) and 8 U of DNase I (Epicentre). These diluted nuclear extracts were pre-cleared using 25 µL of previously blocked Streptavidin Sepharose beads (GE Healthcare) for 2 h at 4°C. Blocking was achieved by incubating the beads with 1 mg.mL−1 of Ultrapure BSA and 0.5 mg.mL−1 of salmon sperm DNA (Invitrogen) overnight at 4°C. The precleared nuclear extracts were recovered, supplemented with 2 µL Riboguard (Epicentre) and incubated for 30 min at room temperature with 500 ng of in vitro synthesized RNAs. 10 µL of blocked beads were then added and incubation was pursued for another 30 min at room temperature. After 4 washes in binding buffer, beads were resuspended in 20 µL binding buffer and 10 µL 4X Laemmli sample buffer (Biorad) supplemented with b-mercaptoethanol. Samples were boiled for 5 min at 95°C before being loaded on a 3–8% NuPAGE Tris-Acetate gel (Invitrogen). Western blots were quantified using ImageJ. Linearity of the EZH2 and SUZ12 western blots was controlled by serial dilutions of an input.
5
Isolation and scRNA-seq of Fetal Liver Progenitors
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For single cell RNA sequencing (scRNA-seq) studies, freshly sorted DAPI−VECadherin−/lowCD45+Gra1−F4/80−Sca1highEPCRhigh cells derived from E13.5 murine fetal liver samples were subject to scRNA-seq experiment. Following the sort (3,050 cells from E13.5), cells were washed and resuspended with PBS containing 0.04% ultrapure BSA (Invitrogen) on ice.
For FL-AKT-EC, confluent ECs in a 6-well plate were cultured in serum-free coculture media (described above) overnight, harvested by treatment with TRypLE Express (Gibco), then resuspended in PBS/10% FBS at 4°C, washed twice and resuspended PBS with 0.04% ultrapure BSA in on ice. A portion of cells (targeting 3,500 cells to load) were subject to downstream scRNAseq assay.
Cell suspensions were loaded into the Chromium Single Cell Chip G (10X Genomics) and processed in the Chromium single cell controller (10X Genomics). The 10X Genomics Version 3.1 single cell 3’ kit was used to prepare single cell mRNA libraries with the Index Kit T Set A, according to manufacturer protocols. Sequencing was performed for pooled libraries from each sample on an Illumina NextSeq 500 using the 75 cycle, high output kit, targeting a minimum of 100,000 reads per cell.
For FL-AKT-EC, confluent ECs in a 6-well plate were cultured in serum-free coculture media (described above) overnight, harvested by treatment with TRypLE Express (Gibco), then resuspended in PBS/10% FBS at 4°C, washed twice and resuspended PBS with 0.04% ultrapure BSA in on ice. A portion of cells (targeting 3,500 cells to load) were subject to downstream scRNAseq assay.
Cell suspensions were loaded into the Chromium Single Cell Chip G (10X Genomics) and processed in the Chromium single cell controller (10X Genomics). The 10X Genomics Version 3.1 single cell 3’ kit was used to prepare single cell mRNA libraries with the Index Kit T Set A, according to manufacturer protocols. Sequencing was performed for pooled libraries from each sample on an Illumina NextSeq 500 using the 75 cycle, high output kit, targeting a minimum of 100,000 reads per cell.
6
Single-cell RNA Sequencing of Immune Cells
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Single-cell RNA sequencing (scRNA-seq) was performed as previously described (10 (link)). Briefly, sorted single-cell suspensions prepared from each sample were washed twice with 0.05% UltraPure BSA (Invitrogen #AM2618) in PBS. For PBMC samples, 6,000 cells were loaded into a 10x Chromium controller instrument along with Chromium Next GEM Single Cell 5′ beads (10x Genomics PN-1000263). Up to four PBMC samples were multiplexed together after being tagged with unique DNA-barcoded antibodies as described above. For tumor-infiltrating lymphocyte (TIL) samples, all sorted cells were loaded, and no multiplexing was performed. After RT-PCR, cDNA was purified, and a library was constructed from each sample using a 10x Library Construction Kit (10x Genomics PN-1000190) following the standard 10x protocol. An additional VDJ-enriched library was created for each sample using a specialized Chromium Single Cell Human TCR Amplification Kit (PN-1000252). Libraries were then sequenced on an Illumina NovaSeq system operated by Azenta/Genewiz generating paired-end 150bp reads.
7
Single-cell transcriptome profiling of PBMCs and TILs
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Sorted single-cell suspensions prepared from each sample were washed twice with 0.05% UltraPure BSA (#AM2618; Invitrogen) in PBS. For PBMC samples, 6,000 cells were loaded into a 10× Chromium controller instrument along with Chromium Next GEM Single Cell 5′ beads (10× Genomics PN-1000263). Up to four PBMC samples were multiplexed together after being tagged with unique RNA tags as described above. For TIL samples, all sorted cells (500-2,000) were loaded, and no multiplexing was performed. After RT-PCR, cDNA was purified, and a library was constructed from each sample using a 10× Library Construction Kit (10× Genomics PN-1000190) following the standard 10× protocol. An additional VDJ-enriched library was created for each sample using a specialized Chromium Single Cell Human TCR Amplification Kit (PN-1000252). Libraries were then sequenced on an Illumina HiSeq system operated by Azenta/Genewiz generating paired-end 150 bp reads.
8
FISH-IF Optimization for Protein Detection
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FISH-IF protocol was adapted from Wu, et al., 2016, Eliscovich, et al., 2017 and the optimized smFISH protocol described above33 (link),42 (link),44 . The procedure is the same as described above except for the differences described below.
Permeabilization was done for 15 minutes at room temperature with 0.1% Triton X 100 and 0.02% UltraPure BSA (Invitrogen #AM2616) in PBSM. Initial 10% pre-hybridization incubation was done with 10% formamide and 0.02% UltraPure BSA in 2X SSC. The 10% hybridization solution contained 1:250 GCN4 Suntag primary antibody along with the secondary probes. After hybridization, two 5 minute washes were performed at 37C with 10% pre-hybridization without BSA. The cells were incubated twice for 20 minutes with 1:800 secondary antibody (Alexa Fluor 647) at 37C.
Permeabilization was done for 15 minutes at room temperature with 0.1% Triton X 100 and 0.02% UltraPure BSA (Invitrogen #AM2616) in PBSM. Initial 10% pre-hybridization incubation was done with 10% formamide and 0.02% UltraPure BSA in 2X SSC. The 10% hybridization solution contained 1:250 GCN4 Suntag primary antibody along with the secondary probes. After hybridization, two 5 minute washes were performed at 37C with 10% pre-hybridization without BSA. The cells were incubated twice for 20 minutes with 1:800 secondary antibody (Alexa Fluor 647) at 37C.
9
Single-cell transcriptomics of aortic cells
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Ascending thoracic aortas were harvested from euthanized P45 Fbn1mgR/mgR and WT mice (n = 3 per genotype), and losartan- and vehicle-treated Fbn1mgR/mgR mice (n = 4 and 3, respectively). Aortic tissues of each genotype and treatment arm were pooled together, cleaned, minced, and digested in Hank’s balanced salt solution (14170112, Gibco) containing elastase (0.5 mg/mL, LS006365, Worthington) and collagenase A (10103578001, Roche) for 1 hour at 37°C and then passed through a 40 μm filter. Single-cell suspensions were incubated with eBioscience 1× RBC Lysis Buffer (00-4333-57, Invitrogen) for 3 minutes and collected in PBS containing 1% UltraPure BSA (AM2616, Invitrogen). After confirming cell count and viability using an EVOS M7000 imaging system (Invitrogen), each sample was immediately processed by the Genomics Core at Icahn School of Medicine at Mount Sinai according to the manufacturer’s instructions (Chromium Next GEM Single Cell 3′ Reagent Kits v3.1, 10× Genomics).
10
Biochemical Reagents for Molecular Assays
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Q5 DNA Polymerase, Q5U DNA Polymerase, Vent (exo-) DNA polymerase, Sulfolobus DNA Polymerase IV, E. coli RNA Polymerase Holoenzyme, RNase If, Thermolabile Proteinase K, Thermolabile USER II Enzyme, T4 DNA Ligase, ET SSB, and Lambda Exonuclease were purchased from New England Biolabs (NEB). UltraPure BSA was purchased from Invitrogen. GreB was a gift from R. Landick (University of Wisconsin, Madison).
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