Four rounds of biopanning using Fab Library E (kind gift of A. Koide) were performed as follows using a protocol adapted from previous studies 40 (link),41 (link). The first round of selection was performed manually using 300 nM of biotinylated K29-linked diubiquitin immobilized onto 250 μL of Streptavidin Paramagnetic Particles (Promega). Following washing with Selection Buffer (SB; 20 mM Hepes, pH 7.5, 150 mM NaCl, 0.5% BSA, 0.05% Tween-20), the beads were incubated with Library E for one hour at room temperature (RT). The beads were then washed three times with SB and used to directly infect log-phase E. coli XL-1 blue cells. Following infection, 2XYT media supplemented with ampicillin and M13 helper phage was added and phage were amplified overnight. To increase the stringency of selection, three additional rounds of sorting were performed in the presence of 1.5-μM of mono-ubiquitin as a competitor in solution. Further, the concentration of K29-linked diubiquitin was reduced each round as follows: 150 nM in round 2, 75 nM in round 3, and 15 nM in round 4. These three rounds were performed at RT semi-automatically using a Kingfisher magnetic beads handler. The overnight-amplified elution pool from each preceding round of selection was used as the input for the following round of sorting.
Streptavidin paramagnetic particles
Manufactured by Promega
Streptavidin Paramagnetic Particles are uniform, superparamagnetic beads coated with the high-affinity protein streptavidin. They are designed for use in a variety of biomolecule separation and purification applications.
Automatically generated - may contain errors
Lab products found in correlation
Magnetic beads handler, by Kingfisher Biotech (2 mentions)
Vent exo polymerase, by New England Biolabs (1 mentions)
Dynabeads, by Abcam (1 mentions)
Ab230356, by Abcam (1 mentions)
Spectramax plus 384 microplate reader, by Molecular Devices (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Mirnaeasy kit, by Qiagen (1 mentions)
5 protocols using streptavidin paramagnetic particles
1
Biopanning for Ubiquitin-Binding Antibodies
2
Microsatellite DNA Enrichment and Amplification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The microsatellite DNA regions of interest were selectively recovered using a Magenesphere magnetic separation kit (Promega) and biotin-labeled probes (GT)10 and (CT)10. After the addition of 50 μL of hybridization solution (12× SSC, 0.1% SDS) together with the probes to the linker-ligated DNA, the mixture (100 μL) was heated to 95 °C for 15 min and then incubated at 60 °C for 12 h. Hybridization was performed using Streptavidin paramagnetic particles (Promega) in accordance with the manufacturer’s instructions. Single-stranded DNA was eluted at 95 °C for 15 min in 100 μL of low-TE buffer containing 10 mM Tris-HCl (pH 8.0) and 0.1 mM EDTA. PCR was performed in mixtures consisting of 10 μL of DNA, 4 μL of 10 μM SNX primer, 0.3 μL of Vent (-exo) polymerase (New England Biolabs), 4 μL of dNTP mix, 5 μL of 10× Thermopol buffer, and 26.7 μL of sterilized distilled water with denaturation at 96 °C for 5 min followed by 40 cycles of 96 °C for 45 s, 62 °C for 1 min, 72 °C for 2 min, and a final elongation step at 72 °C for 5 min.
3
Quantifying m6A Levels in miR-20a-5p
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total RNA content was separated from 1 × 106 cells using the TRIzol reagent (Invitrogen). The RNA m6A level was determined using the m6A RNA Methylation Quantification kit, and the absorbance value was analyzed using the SpectraMax Plus384 microplate Reader (Molecular Device, Sunnyvale CA, USA). Briefly, 150 µg of the total RNA content was dissolved in 500 mL of Rnase-free water and mixed with Biotinylated-Oligo (Promega) at room temperature for 10 min, followed by the addition of Streptavidin-Paramagnetic Particles (Promega). RNA containing polyA+ was separated from the solution using magnetic beads with biotin-streptavidin conjugate. The polyA+ enriched RNA was fragmented using the RNA Fragmentation Buffer (Millipore, Bedford, MA, USA). Dynabeads containing 5 µg of anti-m6A (at a dilution ratio of 1:1000; ab230356; Abcam) were used to bind to RNA fragments containing m6A methylation prior to the elution of RNA fragments from beads and overnight precipitation at 4°C. The enrichment of m6A in miR-20a-5p or pri-miR-20a-5p was detected using the provided primer-probe sets (Bogu Co., Ltd, Shanghai, China).
4
PolyA+ RNA Extraction from Cardiac Tissue
5
Biopanning of Fab Library for K29-linked Diubiquitin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Four rounds of biopanning using Fab Library E (kind gift of A. Koide) were performed as follows using a protocol adapted from previous studies 41, 42 . The first round of selection was performed manually using 300 nM of biotinylated K29-linked diubiquitin immobilized onto 250 µL of Streptavidin Paramagnetic Particles (Promega). Following washing with Selection Buffer (SB; 20 mM Hepes, pH 7.5, 150 mM NaCl, 0.5% BSA, 0.05% Tween-20), the beads were incubated with Library E for one hour at room temperature (RT). The beads were then washed three times with SB and used to directly infect log-phase E. coli XL-1 blue cells. Following infection, 2XYT media supplemented with ampicillin and M13 helper phage was added and phage were amplified overnight. To increase the stringency of selection, three additional rounds of sorting were performed in the presence of 1.5-µM of mono-ubiquitin as a competitor in solution. Further, the concentration of K29-linked diubiquitin was reduced each round as follows: 150 nM in round 2, 75 nM in round 3, and 15 nM in round 4. These three rounds were performed at RT semi-automatically using a Kingfisher magnetic beads handler. The overnight-amplified elution pool from each preceding round of selection was used as the input for the following round of sorting.
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!