mESCs were transfected with FLAG-WT DIS3L2, FLAG-mutant DIS3L21 (link), or empty pFLAG-CMV2 (as mock) vectors. At 48 h after transfection, mESCs were ultraviolet crosslinked, lyzed, and then RNA IP was performed using anti-FLAG M2 Affinity Gel beads (Sigma) as previously described7 (link), and co-precipitated RNAs were isolated, purified, and analyzed by qRT-PCR. Thirty micrograms of FLAG-SRP68 vector (OriGene Technologies, MR204949) was transfected into 107 ESCs in 15-cm dishes of DIS3L2 heterozygote (control) or knockout ESCs in triplicates using Lipofectamine 2000 (Invitrogen) overnight before changing the medium. At 48 h after transfection, cells were harvested without crosslinking and FLAG-SRP68 was precipitated using anti-FLAG M2 Affinity Gel beads (Sigma). Rabbit anti-RPL23a (Proteintech Group; 10 µg antibody per IP) or normal rabbit IgG (Cell signaling) and protein A agarose beads (Roche) were used for IP of ribosomes in control and DIS3L2 knockout ESCs. Co-precipitated RNAs were isolated, purified, and analyzed by qRT-PCR.
Anti flag m2 affinity gel beads
Manufactured by Merck Group
Sourced in United States
The Anti-FLAG M2 affinity gel beads are a laboratory tool used for the purification and detection of proteins tagged with the FLAG epitope. The beads are composed of agarose and are functionalized with the anti-FLAG M2 monoclonal antibody, which binds specifically to the FLAG tag. This allows for the efficient capture and isolation of FLAG-tagged proteins from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Flag peptide, by Merck Group (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Rabbit anti rpl23a antibody, by Proteintech (2 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (2 mentions)
3xflag peptide, by Merck Group (2 mentions)
Pei max, by Polysciences (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Blocked membrane lipid strips, by Echelon Biosciences (2 mentions)
Normal rabbit igg, by Cell Signaling Technology (1 mentions)
Protein a agarose bead, by Roche (1 mentions)
Rabbit anti rpl23a, by Proteintech (1 mentions)
Protein g sepharose beads, by Merck Group (1 mentions)
Cfx384 touch real time pcr detection system, by Bio-Rad (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Protein g sepharose, by Thermo Fisher Scientific (1 mentions)
Power sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Benzonase, by Merck Group (1 mentions)
29 protocols using anti flag m2 affinity gel beads
1
RNA Immunoprecipitation of FLAG-Tagged DIS3L2 and SRP68 in mESCs
2
Co-Immunoprecipitation of FLAG-BirA* Fusions
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For co‐immunoprecipitation of FLAG‐BirA* fusions, one 10‐cm dish of the respective HEK293 stable lines was incubated with Tet (1 μg/ml) for 24 h. After incubation, cells were washed with cold PBS and harvested. Cells were subsequently frozen at −80°C or lysed immediately in lysis buffer (50 mM HEPES pH 8, 100 mM KCl, 2 mM EDTA, 10% glycerol, 0.1% NP‐40, 1 mM DTT, protease inhibitors (Roche)) for 30 min on ice. Lysates were frozen for 5 min on dry ice, then thawed and centrifuged for 20 min at 16,000 g at 4°C. Supernatants were incubated with anti‐FLAG M2 affinity gel beads (MilliporeSigma) for a minimum of 3 h at 4°C (prior to incubation with beads, a fraction of supernatants (inputs) were saved). After incubation, beads were pelleted and washed with lysis buffer. Laemmli buffer was added, and samples (inputs and IPs) were boiled 5 min at 95°C in preparation for SDS–PAGE. Proteins were transferred to PVDF membranes and probed using anti‐FLAG to detect the FLAG‐BirA* fusions, and anti‐TPGS1 or other satellite antibodies for the endogenous protein (antibodies listed in Table EV11 ).
For the co‐IPs using drug‐treated samples, FLAG‐BirA* only and FLAG‐BirA*‐PCM1 cells were grown in the presence of Tet and centrinone or DMSO for 7 days, and then processed as described above. In the case of microtubule disruption, nocodazole was added to the Tet‐induced cells 3 h before harvesting.
For the co‐IPs using drug‐treated samples, FLAG‐BirA* only and FLAG‐BirA*‐PCM1 cells were grown in the presence of Tet and centrinone or DMSO for 7 days, and then processed as described above. In the case of microtubule disruption, nocodazole was added to the Tet‐induced cells 3 h before harvesting.
3
Triton X-100 Lysis and Immunoprecipitation
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Cellular lysates were prepared using a Triton X-100 lysis buffer, consisting of 50 mM tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM N-ethylmaleimide, 2 mM Na3VO4, 20 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 1× protease inhibitor cocktail and 0.5% (v/v) Triton X-100. Cellular lysates were clarified by centrifugation at 14,000g at 4°C for 15 min. The resulting supernatants were subjected to immunoprecipitation and immunoblot analysis with the corresponding antibodies. For immunoprecipitation, proteins (1 mg) were pre-incubated with Protein G Sepharose beads (MilliporeSigma) for preclearing and further incubated with 1 to 2 μg of the corresponding antibodies or anti-Flag M2 affinity gel beads (MilliporeSigma) overnight at 4°C. The immunocomplexes were collected with Protein G Sepharose beads followed by centrifugation at 3000g at 4°C for 2 min. Proteins were eluted from the beads by addition of 2× protein sample buffer, denatured by boiling, separated on SDS–polyacrylamide gel electrophoresis, and subjected to immunoblot analysis. Indicated antibodies were used. Enhanced chemiluminescence was used to detect specific bands using standard methods. The relative band intensity was measured using the ImageJ imaging software.
4
RNA Immunoprecipitation and qPCR Analysis
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RNA IP was performed as previously described (Akef et al. 2013 (link); Lee et al. 2015 (link)). FLAG-TAP plasmid (inserted in p3xFlag-CMV-10 vector, as previously described [Lykke-Andersen et al. 2001 (link)]) was cotransfected with the various reporter plasmids. For UAP56 pulldowns, cell lysate from transfected cells was incubated for 10–14 h with either rat anti-UAP56 antibodies (Yamazaki et al. 2010 (link)) or rat preimmune serum prebound to protein G sepharose (Invitrogen). For FLAG-TAP pulldowns, cell lysate from transfected cells was incubated with ANTI-FLAG M2 Affinity Gel beads (A2220, Sigma-Aldrich) or protein A beads for 2.5 h. cDNA was synthesized using SuperScript III (Invitrogen) according to the manufacturer's protocol. qPCR was performed by mixing the cDNA with Power Sybr Green Master Mix (Invitrogen) and the reaction was run on a CFX384 Touch Real Time PCR Detection System (Bio-Rad). The efficiency of the IP reaction was confirmed by separating the cell lysate and immunoprecipitates by SDS–PAGE, transferring the proteins to nitrocellulose and immunoblotting for UAP56 using rabbit anti-UAP56 antibodies (Sigma).
5
Immunoprecipitation of FLAG-tagged Evectin-2
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COS-1 cells grown on 100-mm dishes were transfected with FLAG-tagged evectin-2 (WT, PPPA, or ΔPPPY) or empty vector (pcDNA3-FLAG) using Lipofectamine 2000 (Invitrogen). At 24 h after transfection, the cells were lysed in 1 ml of immunoprecipitation buffer (25 mM HEPES-KOH pH 7.33, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100, cOmplete EDTA-free Protease inhibitor cocktail (Roche)). The cell lysates were centrifuged at 15,000×g for 20 min at 4 °C, and the resultant supernatants were incubated for 2 h at 4 °C with anti-FLAG M2 affinity gel beads (Sigma). The beads were washed three times with immunoprecipitation buffer. Immunoprecipitated proteins were analyzed by western blotting.
6
Transient Expression and Immunoprecipitation of ZMYND8
7
KLF4 Ubiquitination Assay using Mule
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293T cells overexpressing Mule or MuleC4341A (see below) were grown to near-confluence. Lysates were prepared using standard methods and incubated with Anti-Flag M2 affinity gel beads (Sigma) at 4 °C with rotation for 2 h. Flag-Mule or Flag-MuleC4341A was eluted by adding 3 × Flag peptide (Sigma) at a final concentration of 100 μg ml−1 in 1 × ubiquitination buffer (50 mM Tris pH 7.4, 2 mM ATP, 5 mM MgCl2 and fresh 2 mM DTT) to the washed beads. Tubes were rotated at 4 °C for 5–10 min. For the KLF4 ubiquitination assay, 0.1 ng human E1, 0.4 ng Ubc5/7 E2, 3 μl of eluted Mule or MuleC4341 protein, 2 mM ubiquitin and 2 mM fresh ATP were mixed in ubiquitination buffer to a final volume of 20 μl. The reaction mixtures were incubated in a PCR machine at 30 °C for 90 min, when 5 μl SDS–PAGE buffer (5 × ) was added to stop the reaction. Ubiquitinated proteins were separated by 4–12% SDS–PAGE and detected by immunoblotting with anti-ubiquitin (FK2; Enzo Life Science) or anti-KLF4 (GeneTex) Ab.
8
RNA Immunoprecipitation of Dis3l2 in mESCs
9
Crosslinking and Immunoprecipitation of EPEC Proteins
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50 mL of DMEM without calcium or the same medium added with 1.8 mmol/L CaCl2 was inoculated with O/N cultures of the EPEC sepL‐3FLAG cesL‐2HA::km or EPEC cesL‐2HA::km strains. Cell cultures were grown under static conditions at 37°C in a 5% CO2 atmosphere. At an OD600 of 1, cultures were harvested by centrifugation, cell pellets were washed with HEPES 20 mmol/L, NaCl 250 mmol/L pH 7.4, resuspended in the same buffer and lysed by sonication. Cell lysates were incubated with Triton X‐100 [0.1% (v/v)] for 15 min and then cleared by centrifugation at 19,800g for 30 min. Supernatants were carefully collected and the crosslinker dithiobis succinimidyl propionate was added to a final concentration of 1 mmol/L. After 1 hr incubation at room temperature, the crosslinking reaction was stopped by the addition of 20 mmol/L Tris pH 7.5. Cross‐linked samples were mixed with 40 μl of preequilibrated ANTI‐FLAG M2 Affinity Gel beads (SIGMA) and incubated O/N at 4°C with shaking. SepL‐3FLAG coupled beads were centrifuged at 13,500g for 2 min and washed three times with HEPES 20 mmol/L, NaCl 250 mmol/L pH 7.4. Finally, the beads were resuspended in 20 μl of SDS‐PAGE sample buffer, containing 2 μl of β‐mercaptoethanol and boiled for 5 min. Protein samples were resolved by SDS‐PAGE and analyzed by immunoblotting as described above.
10
Daple Protein Expression and Purification
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Daple FL, Daple CT, Daple NT, and Daple ΔCT were amplified using PCR from a mouse brain cDNA library and inserted into the pEGFP-C1-Flag vector, which was constructed by the insertion of a Flag-tag into the pEGFP-C1 vector (6084-1; Clontech). We then modified EGFP to mEGFP with an A206K mutation to obtain the mEGFP-Daple plasmids. HEK293T cells in 10-cm dishes were transfected with 6 µg mEGFP-Daple-Flag plasmids using PEI MAX (Polysciences) according to the manufacturer’s instructions. Cells were washed three times with ice-cold HBS and then scraped with 200 µl lysis buffer (250 mM NaCl, 10% sucrose, 1 mM MgSO4, 20 mM Hepes, pH 7.5, and a protease inhibitor cocktail [03969; Nacalai Tesque]). The resuspended cells were then disrupted using a sonicator and centrifuged at 20,400 × g at 4°C for 30 min. The supernatant was incubated with 20 µl anti-Flag M2 affinity gel beads (A2220; Sigma-Aldrich) at 4°C for 18 h. Following incubation, the beads were washed four times with ice-cold HBS and then eluted with the 3X-FLAG peptide (F4799; Sigma-Aldrich) in ice-cold HBS containing the protease inhibitor cocktail (03969; Nacalai Tesque) and 1 mM DTT (Y00147; Invitrogen) following the manufacturer’s instructions.
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