Cells were treated with FLAG–H114R ANG, and then washed with cold PBS. Cellular contents were then crosslinked by using 254-nm light at 1500 J/cm2. Cells were lysed in IP buffer (20 mM HEPES–KOH buffer, pH 7.5, containing 250 mM NaCl, 1 mM EDTA, 1% v/v NP-40, 10% v/v glycerol, and a protease-inhibitor cocktail) for 20 min at 4°C, and the cell lysate was subjected to centrifugation. The clarified lysate was treated with 500 pM RNase A and 2 μl of 2 units/μl DNase I (Invitrogen) for 10 min at 37°C, and then incubated with α-FLAG magnetic beads (Sigma Chemical). The beads were washed and then eluted three times with 150 ng/μl 3× FLAG peptide (APExBIO). The combined eluates were split into two portions: one for an immunoblot and the other for RT-PCR to identify co-precipitated RNAs. To determine its sequence, the PCR product was incorporated into a TOPO®TA vector (Invitrogen), which enables use of the M13 forward primer.
α flag magnetic beads
Manufactured by Merck Group
α-FLAG magnetic beads are a laboratory tool used for the purification and detection of FLAG-tagged proteins. They consist of magnetic particles coated with an antibody that specifically binds to the FLAG peptide sequence, allowing for the efficient isolation and enrichment of FLAG-tagged proteins from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Topo ta vector, by Thermo Fisher Scientific (1 mentions)
Flag peptide, by Apexbio (1 mentions)
Elution buffer, by Thermo Fisher Scientific (1 mentions)
Ip buffer, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor tablet, by Roche (1 mentions)
Phosstop phosphatase inhibitor tablet, by Roche (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Imagequant tl software, by GE Healthcare (1 mentions)
4 protocols using α flag magnetic beads
1
RNA-Protein Interactome Identification
2
Immunoprecipitation of CLPP variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three independent Clpp−/− MEF lines were transfected with each empty vector (NEG), CLPP-WT-FLAG (WT) and a catalytically inactive version with Ser149 mutated to Ala, CLPP-TRAP-FLAG (TRAP) (13 (link)) using the Nucleofector (Lonza, Basel, Switzerland) electroporation kit according to the manufacturer instruction. 72h after transfection, 80% confluent cells were harvested with trypsin, washed twice with PBS and lysed for 45 min in 300 μl IP buffer (Thermo Fisher Scientific, Schwerte). Samples were incubated with 30 μl α-FLAG magnetic beads (Sigma) over night at 4 °C on a rotating wheel. On the following day, beads were washed 4 times with IP buffer and bound proteins were eluted in 70 μl Elution buffer (Thermo Fisher Scientific). Samples were neutralized with 10 μl 1 m Tris/HCl pH 7.5, snap-frozen and stored at −80 °C. 1% of each Lysate (L), unbound proteins (F), first washing solution (W) and 10% of the Elution fractions (E) were used for SDS-PAGE controls. Samples were prepared for proteomic analysis as previously described (13 (link)).
3
Ubiquitin Co-Immunoprecipitation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were collected in lysis buffer (50mM Tris-HCl, 150mM NaCl, 5mM EDTA, 1% NP-40, 20mM MG132, 1mM PMSF, 10mM Iodoacetamide, 1mM 1,10-Phenanthroline monohydrate, Roche Complete protease Inhibitor tablets, Roche Phos-Stop phosphatase inhibitor tablets) and centrifuged to isolate protein. Protein concentration was measured using Bradford Assay and a 1 mg/ml protein solution was made. For Ub co-immunoprecipitation experiments, cells were transiently transfected with HA-Ub plasmid 24 hours before lysing. 1ml of 1 mg/ml solution was added to α-FLAG magnetic beads (Sigma). For whole cell lysates, Laemmli buffer was added and samples were put at 65°C for 10 min. Co-IPs were incubated at 4°C for 1 h with rotation, washed 3 times with wash buffer (25mM Tris pH 7.5, 2.5% glycerol, 150mM NaCl), and eluted by incubation with FLAG peptide for 30 min at 4°C twice. Protein samples were run on 12% polyacrylamide gels, transferred onto PVDF membrane, and imaged after antibody incubation on the LiCor Odyssey CLx Infrared imager.
4
Immunoblotting and Immunoprecipitation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells grown in a 10-cm dish were lysed with 1 ml of M-PER mammalian protein extraction reagent (Pierce) containing a protease-inhibitor cocktail. Protein (∼30 μg) was separated by SDS–PAGE (Bio-Rad), and the resulting gel was subjected to immunoblotting. Densitometry data were analyzed with ImageQuant TL software (GE Healthcare Life Sciences). For immunoprecipitation experiments, cells were lysed with IP buffer containing protease- and phosphatase-inhibitor cocktails. Clarified lysates were incubated with α-FLAG magnetic beads (Sigma Chemical), and the beads were washed three times. Samples were eluted with 30 μl of SDS gel-loading dye and processed further for immunoblotting.
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!