PAR-CLIP was performed as previously described (Hafner et al. 2010 (link)) with minor modifications. Briefly, a total of 400 million cells were used for each experiment. Each plate was treated with 4-Thiouridine (Sigma-Aldrich) at a final concentration of 100 µM 14 h prior to UV-crosslinking with 0.15 J/cm2 of 365-nm UV light with a Stratalinker UV Crosslinker (Stratagene). Cells were then scraped, lysed, and digested with RNase T1 (Fermentas) to a final concentration of 1 unit/µL. AGO immunoprecipitation of the lysate was then performed using Dynabeads Protein G magnetic particles (Invitrogen) and AGO antibody (Sigma) at a final concentration of 0.05 µg/µL. A second RNase T1 treatment was then performed using a final concentration of 10 units/µL. The RNA segments were then radiolabeled using 32P-γATP to a final concentration of 0.5 µCi/µL and T4 PNK to a final concentration of 1 unit/µL, and samples were then resuspended in 70 µL SDS-PAGE loading buffer, and SDS-PAGE was performed. The gel bands corresponding to AGO were cut for each sample and electroelution of the crosslinked RNA–protein complexes was then performed. Recovery of crosslinked target RNA fragments was then performed using phenol chloroform and ethanol extraction.
Dynabeads protein g magnetic particles
Manufactured by Thermo Fisher Scientific
Dynabeads Protein G magnetic particles are designed for the purification and isolation of antibodies and other proteins. The particles are superparamagnetic, allowing for easy separation using a magnet. Protein G, a bacterial cell wall protein, is covalently coupled to the surface of the particles, providing a high-affinity binding site for the Fc region of immunoglobulins.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase k, by Thermo Fisher Scientific (2 mentions)
Rnase t1, by Thermo Fisher Scientific (1 mentions)
4 thiouridine, by Merck Group (1 mentions)
Rnase t1, by Merck Group (1 mentions)
Protease inhibitor mixture, by Roche (1 mentions)
Ago2 antibody, by Fujifilm (1 mentions)
Mouse igg, by Santa Cruz Biotechnology (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
4 protocols using dynabeads protein g magnetic particles
1
PAR-CLIP Protocol for Crosslinked RNA-Protein Complexes
2
Protein Immunoprecipitation Optimization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were washed twice with PBS and linked with 12000 J/cm2 UV light for 2 min. The lysate is treated with 30% amplitude ultrasound for 5 minutes, 3 seconds pulse, followed by 6 seconds rest period. 50μl Dynabeads Protein G magnetic particles (Invitrogen) were resuspended in 500μl lysis buffer. The beads were precipitated by magnet and then suspended again in the clear lysate and incubated at 4°C for 2h. All beads were resuspended in lysis buffer and subjected to western blots.
3
Chromatin Immunoprecipitation Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were washed twice with ice-cold PBS and cross linked with UV 12000 J/cm2 for 2 min in PBS. The cells were incubated on ice in modified RIPA lysis buffer (150 mM NaCl, 50 mM Tris pH 8.0, 1% Nonidet P-40, 0.5% deoxycholate, and a protease inhibitor mixture (Roche Applied Science, Mannheim, Germany)) for 20 min and rotated at 4°C for 10 min. Then, lysate was sonicated typically for 5 min at 30% amplitude, 3 sec pulses followed by 6 sec rest period. The lysates were clarified by centrifugation for 20 min at 14,000 rpm at 4°C. Fifty (50 μl) of Dynabeads Protein G magnetic particles (Invitrogen) were resuspended, 500 μl of lysis buffer. Then, 1.5 μg of respective antibodies was added to the beads and incubated on a rotating wheel at room temperature for 30 min. Beads were precipitated by magnet and finally resuspended again in cleared cell lysates and incubated for 2 h at 4°C. In the last step, the beads were resuspended in 50 μl of lysis buffer and subjected to Western blots.
4
UV-Crosslinking and RNA Immunoprecipitation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dissociated‐EB6 were resuspended in PBS (without Ca2+ and Mg2+) and plated in dishes (10 × 10 cm2). Cells were UV cross‐linked with 4,000 µJoules/cm² energy using a stratalinker and centrifuged at 200 g for 5 min at 4°C. Cell pellets were resuspended in 3 volumes of NP40 lysis buffer pH 7.5 (50 mM Hepes‐KOH; 150 mM KCl; 2 mM EDTA; 1 mM NaF; 0.5% NP40; 0.5 mM DTT; 1× PIC) and incubated on ice for 10–15 min followed by centrifugation at 18,000 g for 10 min at 4°C. Resulting cellular lysates were incubated (overnight on a rotating wheel, at 4°C) with 30 µl of Dynabeads Protein G magnetic particles (Invitrogen) preincubated with either 10 μg of Ago2 Antibody (018‐22021, Wako) or mouse IgG (sc‐2025, Santa Cruz). Beads were washed with a High‐Salt buffer (50 mM Hepes‐KO; 500 mM KCl; 0.5 mM DTT; 0.05% NP40). Before RNA extraction, 1/5 of the cell lysate was heated for 5 min at 95°C, and the supernatant was collected and resuspended in Protein elution buffer (4× Laemmli sample buffer [BioRad]) with DTT 50 mM and analyzed by Western blot. RNA fraction was treated with Proteinase K (AM2546, Thermo Fisher Scientific) for 30 min at 50°C; the samples were then placed for 10 min at 95°C, and finally, the RNA was extracted using miRNeasy Mini Kit with on‐column DNAse treatment, according to the manufacturer’s instructions (Qiagen).
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!