For 3xFLAG-Ctp IP-MS, 1 × 107 OSCs were seeded in 10 cm dishes, and transfected with 20 µg 3xFLAG- fusion expression plasmid (Xfect) the following day. Cells were harvested 48 hr post-transfection and lysed in IP Lysis Buffer (Pierce) supplemented with protease inhibitors (Roche) for 45 min at 4°C. After centrifugation at 16,500 g for 10 min at 4°C, lysates (with 5% saved as input) were incubated with 50 µl anti-FLAG M2 magnetic agarose beads (Sigma M8823) overnight at 4°C. Beads were washed three times for 15 min in IP Lysis Buffer, twice in ice-cold PBS and twice in 100 mM ammonium bicarbonate before being submitted for mass spectrometry analysis. For the 3xFLAG-Panx IP-MS, 1 × 107 OSCs were nucleofected with 5 µg 3xFLAG- fusion expression plus 2 µl siRNA against endogenous Panx. Cells were harvested 48 hr post transfection and fractionated according to the above protocol. After sonication, the nuclear fraction was additionally treated for 30 min with Benzonase (50 U/ml) at 4°C followed by centrifugation. Nuclear lysate was incubated with 50 µl anti-FLAG M2 magnetic agarose beads (Sigma M8823) overnight at 4°C, washed three times for 15 min in LB3, twice in ice-cold TBS (pH 8.0) and twice in 100 mM ammonium bicarbonate before being submitted for mass spectrometry analysis.
Anti flag m2 magnetic agarose beads
Manufactured by Merck Group
The Anti-FLAG M2 Magnetic Agarose Beads are a laboratory product designed for the purification and detection of proteins tagged with the FLAG epitope. The beads are composed of agarose and contain the Anti-FLAG M2 antibody, which binds to the FLAG tag with high affinity. The magnetic properties of the beads allow for easy separation and washing of the bound protein during the purification process.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
3xflag peptide, by Merck Group (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Nupage sample buffer, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Thermo Fisher Scientific (1 mentions)
7 protocols using anti flag m2 magnetic agarose beads
1
3xFLAG-Ctp and Panx IP-MS Protocols
2
Immunoprecipitation of Nematode RNAs
3
Immunoprecipitation of Nematode RNAs
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Synchronous population of 40,000 worms were harvested at 48 hours post hatching and suspended in the extraction buffer (50 mM HEPES pH7.5, 300 mM NaCl, 5 mM MgCl2, 10% Glycerol, 0.25% NP40, protease inhibitor cocktails (Fermentas)) followed by 30 strokes using a metal dounce on ice. Crude protein extracts were spun at 12,000 rpm at 4°C for 10 minutes. Protein concentration was quantified by the Bradford assay, and 1 mg of protein extract was incubated with 15 μl of packed Anti-FLAG M2 Magnetic Agarose Beads (Sigma M8823) for 1 hour at 4°C. After four washes with extraction buffer, the immunoprecipitated protein was eluted twice with 100 μL of 3XFLAG peptide (Sigma F4799) for 30 minutes at 4°C. Next, the eluate was incubated with 750 μL of TRI Reagent (MRC, Inc.) to extract the immunoprecipitated RNAs. 750 μL of TRI Reagent (MRC, Inc.) was also added to 10% of protein extract before the immunoprecipitation (Input). The extracted RNAs were then analyzed by RT-qPCR to quantify mRNAs or used to clone small RNAs.
4
Worm Proteome Extraction and Co-IP
5
Worm Proteome Extraction and Co-IP
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Synchronous population of 120,000 (for CSR-1, PRG-1 IPs) or 40,000 (for WAGO-1 IPs for mass spectrometry) worms were harvested at 48 hours post hatching and suspended in the extraction buffer (50 mM HEPES pH7.5, 300 mM NaCl, 5 mM MgCl2, 10% Glycerol, 0.25% NP40, protease inhibitor cocktails (Fermentas)) followed by 30 strokes using a metal dounce on ice. Crude protein extracts were centrifuged at 12,000 rpm at 4°C for 10 minutes. Protein concentration was quantified by the Bradford assay, and 2 mg of protein extract was incubated with 15 μl of packed Anti-FLAG M2 Magnetic Agarose Beads (Sigma M8823) for 1 hour at 4°C. After four washes with the extraction buffer the beads were resuspended with 2X NuPAGE LDS Sample Buffer (thermofisher) for Co-IP experiments or washed twice with 100 μL of 25 mM NH4HCO3 for mass spectrometry. For the Co-IP experiments shown in Fig. 4b and Extended Data Fig. 4e the total protein extract from piwi mutant and control strains were normalized on the level of CSR-1 protein to avoid differences in the total germline proteins due to the reduced germline tissue in piwi mutant.
6
Affinity Purification of Protein Complexes
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Four × 106 OSCs were nucleofected with HA- and 3xFLAG-tagged constructs (5 µg total) and harvested for coIP analysis 48 hr post-transfection. Cell pellets were lysed in 250 µl IP Lysis Buffer (Pierce) supplemented with complete protease inhibitors (Roche) for 30 min at 4°C, and then spun at 21,100 g for 10 min at 4°C. Total protein was diluted to 1 ml with IP Lysis buffer and 5% was saved as input. The rest was incubated with 25 µl anti-FLAG M2 magnetic agarose beads (Sigma M8823) overnight at 4°C. Unbound fractions were collected, and the beads were washed in TBS 3 × 15 min at 4°C. Proteins were eluted from the beads in 20 µl 2X NuPAGE sample buffer (Invitrogen) by boiling at 90°C for 3 min prior to western blot analysis.
7
Immunoprecipitation and RNA Extraction
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The synchronous population of 80,000 λN::3xflag::ha::csr-1 or λN::3xflag::mCherry worms were collected 44 h after hatching and suspended in extraction buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 5 mM MgCl2, 10% glycerol, 0.25% NP-40, Halt protease inhibitor cocktails (Thermo Fisher Scientific), Samples were treated by at least 50 strokes using a metal dounce on ice and crude protein extracts were centrifuged at 12,000 r.p.m. at 4 °C for 10 min. Protein concentration was quantified using the Bradford assay, and 1 mg of protein extract was incubated with 15 μl of packed anti-Flag M2 magnetic agarose beads (Sigma, M8823) for 2 at 4 °C. After four washes with extraction buffer, 1mL of TRI reagent (Invitrogen) was directly added to the magnetic beads to extract the immunoprecipitated RNAs. 1mL of TRI reagent was also added to 10% of protein extract before the IP (input). The resulting isolated RNA was analyzed using RT–qPCR to quantify mRNA levels.
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