Proteins were incubated overnight at 4°C or for 2 h at 20°C at a concentration of 50 μg/ml with m7GTP-agarose or blank agarose (both Jena Bioscience), respectively, in a buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol, and 0.005% Tween 20. Agarose beads were washed extensively with the mentioned buffer and SDS sample buffer was added to the beads for subsequent SDS-PAGE analysis.
M7gtp agarose
Manufactured by Jena Biosciences
Sourced in United States
M7GTP-agarose is an affinity chromatography resin designed for the purification of m7GTP-binding proteins, such as eukaryotic translation initiation factors. It consists of 7-methylguanosine-5'-triphosphate (m7GTP) covalently coupled to agarose beads.
Automatically generated - may contain errors
Lab products found in correlation
M7gtp agarose, by Thermo Fisher Scientific (2 mentions)
M7g 5 ppp 5 g cap analog, by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Gtp sepharose, by Jena Biosciences (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Protease inhibitor tablet, by Roche (1 mentions)
U2os cell, by American Type Culture Collection (1 mentions)
8 protocols using m7gtp agarose
1
Protein Capture and Analysis
2
Cap-binding Protein Interaction Assay
3
Cap-binding Protein Interaction Assay
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To analyze the interaction of METTL3 to cap-binding protein complex, cells were lysed using NET2 buffer and total cell extracts were incubated with m7GTP-Agarose (Jena Bioscience, AC-155S) for 2 hours at 4°C. Then, the beads were washed for five times and suspended in SDS sample buffer. The eluted samples were analyzed by Western blot. Where indicated, 75 μM of m7G(5’)ppp(5’)G Cap Analog (Ambion, AM8048) was added into the sample and incubated with m7GTP-Agarose.
4
LARP1 Identification via m7GTP Pulldown
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Jurkat cells were cultured in RPMI media, supplemented with non-essential amino acids (Gibco) and sodium pyruvate (Gibco). Jurkat cell pellets were lysed using Buffer B (10 mM HEPES pH 7.4, 1 mM MgCl2, 10 mM NaCl, 50 mM NaF, 0.5% NP-40). Lysates were pre-cleared with Protein A-Sepharose beads (Generon, PCA-125) for 30 min at 4°C, rotating. Lysate were split into equal parts, each incubated with 40 ul m7GTP-Agarose (Jena Bioscience) or GTP-Sepharose for 2 h at 4°C. Beads were washed with Buffer B. After washes, beads were re-suspended in LDS sample buffer (Novex) with 0.1 M DTT diluted in Buffer B. m7GTP and GTP pulldown samples were resolved on the SDS-PAGE gels with SDS Running Buffer (25 mM Tris, 250 mM glycine, 0.1% SDS), then transferred to PVDF membranes, which were probed with rabbit anti-LARP1 antibody (ProteinTech).
5
m7GTP-Agarose Pulldown Assay
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50 μl of m7GTP-agarose (Jena Bioscience catalogue # AC-155L) per pulldown was gently centrifuged, storage buffer aspirated and beads resuspended in an equal volume of m7GTP lysis buffer (50 mM MOPS/KOH pH7.2, 50 mM NaCl, 2 mM EGTA, 5 mM EDTA, 7 mM β-mercaptoethanol, 50 mM β-glycerophosphate, 50 mM NaF, 1% (v/v) IGEPAL, 1% (v/v) Triton-X-100, protease inhibitor tablet (Roche Applied Sciences catalogue # 11836170001)). 600 μl of each normalized lysate was incubated with 50 μl of m7GTP-agarose (pre-equilibrated in lysis buffer). Pulldowns were performed for 1 h at 4°C with end over end rotation. Beads were gently pelleted for 20 s at 4000 rpm, the supernatant removed and 1 ml wash buffer added ((50 mM MOPS/KOH pH 7.2, 50 mM NaCl, 2 mM EGTA, 5 mM EDTA, 7 mM β-mercaptoethanol, 50 mM β-glycerophosphate, 50 mM NaF). The wash step was repeated twice and beads were resuspended in 70 μl sample buffer and heated at 95°C for 5 min prior to running on SDS-PAGE gels and western blotting.
6
Measuring eIF4F Complex Displacement
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eIF4F was assembled onto m7GTP-agarose (Jena Bioscience) as previously described (9 (link)). Assembled eIF4F complexes were challenged with 100 pmol of indicated RNA for 2 h at 4°C while tumbling. Displaced proteins were washed away with NP-40 lysis buffer (10 mM Tris [pH 8.0], 100 mM NaCl, 0.5% NP-40, 1 mM EDTA) and bound proteins were eluted with 60 μl of 1X SDS-Loading dye. eIF4F displacement was assessed by analyzing eIF4G and eIF4E by western blotting.
7
Affinity Purification of eIF4F Complex
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Cell lysates were prepared from U2OS cells (ATCC, USA) by lysis with NP-50 Lysis buffer (10 mM Tris (pH 8.0), 100 mM NaCl, 0.5% NP-40) and eIF4F was assembled onto m7GTP agarose (Jena Bioscience) by tumbling for 2 h. Assembled complexes were further purified by washing with NP-40 lysis buffer to remove unbound proteins25 (link). Assembled eIF4F complexes were challenged with 100 pmol of indicated RNA for 2 h at 4 °C while tumbling. Displaced proteins were washed away with NP-40 Lysis buffer and bound proteins were eluted with 60 μL of 1X SDS Loading dye. eIF4F displacement was assessed by analyzing eIF4G and eIF4E by western blotting.
8
Purification of eIF4E-eIF4G Complexes
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Highly purified eIF4F was purified from rabbit reticulocytes as previously described (16 (link)). A graphical representation of the purification schema is shown in Figure 1A . For purification of eIF4E:eIF4G complexes from E. coli, pET28-eIF4E, pET28-eIF4G (90–651), or pET28-eIF4G (90–1129) were transformed into Rosetta2 pLysS cells, grown in 2XYT media until OD reached 0.6 and then induced with 1 mM IPTG for 4 h. Aliquots of bacteria were spun down (1 ml for eIF4E and 10 ml for eIF4G) and frozen at –80°C. For purification, frozen aliquots were thawed on ice and resuspended in 20 mM Tris [pH 8.0], 150 mM NaCl, 0.5 mg/ml lysozyme. Cells were further disrupted by sonication and lysate was clarified by centrifugation. 10% NP-40 was added to supernatant to a final concentration of 0.5%. Individual lysates or combined lysates were incubated with m7GTP-agarose (Jena Biosciences) for 2 h and washed 3× with 20 mM Tris [pH 8.0], 150 mM NaCl, 0.5% NP-40. Cap competition assays were performed with small RNAs as described previously (4 (link)).
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