Protocols to isolate p53 complexes from GBM cells (U87MG line) have been previously described.13 (link) Briefly, using the NE-PER kit (Thermo Fisher Scientific), cytoplasmic and nuclear fractions were separated from GBM cell pellets (~100 μL/pellet). The nuclear fraction was incubated with Ni–NTA (nickel–nitrilotriacetic acid) agarose beads (Qiagen) for 1 h at 4 °C. Beads were then washed with five bed volumes of 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-salt buffer (20 mM HEPES, (pH 7.2), 140 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, and 5 mM imidazole). Complexes were eluted with the same HEPES buffer, supplemented with 60 mM imidazole. Fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) denaturing gels through SimplyBlue SafeStain (Invitrogen) or western blot.13 (link),14 Western blot analysis used antibodies against p53 (DO-1; Santa Cruz Biotechnology, sc-126) to detect complexes.
Agarose beads
Manufactured by Qiagen
Sourced in United States
Agarose beads are a type of chromatography resin used for the separation, purification, and analysis of biomolecules such as proteins, nucleic acids, and other macromolecules. They provide a porous matrix that allows for the efficient binding, washing, and elution of target molecules. Agarose beads offer a versatile and widely-used platform for a variety of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ni2 nta, by Qiagen (2 mentions)
Sc 126, by Santa Cruz Biotechnology (1 mentions)
Ne per kit, by Thermo Fisher Scientific (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
Hygromycin, by Merck Group (1 mentions)
Zeocin, by Merck Group (1 mentions)
Paromomycin, by Merck Group (1 mentions)
Dna ladder, by New England Biolabs (1 mentions)
Protein ladder, by Thermo Fisher Scientific (1 mentions)
Borrelidin, by Abcam (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Hydroxylamine ha, by Thermo Fisher Scientific (1 mentions)
Ni nta silica beads, by Qiagen (1 mentions)
Tmt 16 reagents, by Thermo Fisher Scientific (1 mentions)
Dmso hplc grade, by Merck Group (1 mentions)
Tris 2 carboxyethyl phosphine hydrochloride tcep hcl, by Thermo Fisher Scientific (1 mentions)
Milli q system, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Urea, by Merck Group (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
4 protocols using agarose beads
1
Isolation of p53 Complexes from GBM Cells
2
Purification of LdThrRS Enzyme
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All restriction enzymes and DNA ladders were acquired from New England Biolabs (NEB) (MA, USA). The expression vector pET30a was obtained from Novagen (Germany). Ni2+-NTA (nitrilotriacetic acid) agarose beads were obtained from Qiagen (USA). Hygromycin, paromomycin and zeocin were acquired from Sigma Aldrich (Germany). Protein ladders were obtained from Fermentas. Borrelidin was purchased from Abcam (Cambridge, UK). The in vitro tRNA transcription kit was obtained from Invitrogen (CA, USA). The anti-LdThrRS antibody was commercially synthesized in rabbits by Merck (Germany).
3
Quantitative Proteomics Sample Preparation
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Urea, dithiothreitol (DTT), iodoacetamide (IAA), iron chloride, triethylammonium bicarbonate (TEABC), ethylenediaminetetraacetic acid (EDTA), ammonia phosphate (NH4H2PO4), trifluoroacetic acid (TFA), formic acid (FA), acetonitrile (ACN), n-Dodecyl β-D-maltoside (DDM), protease, phosphatase inhibitor, DMSO (HPLC grade), and Phosphate-Buffered Saline (PBS) were obtained from Sigma (St. Louis, MO). The Ni-NTA silica beads and agarose beads were obtained from Qiagen (Hilden, Germany) and the EmporeTM extraction C18 disks were obtained from 3 M (St. Paul, MN). TMT-16 reagents were purchased from Thermo Fisher Scientific (Waltham, MA). Water was processed using a Millipore Milli-Q system (Bedford, MA). Polypropylene microwell chip with 2.2-mm wells diameter was manufactured on polypropylene substrates by Protolabs (Maple Plain, MN). Tris(2-carboxyethyl) phosphine hydrochloride (TCEP-HCl), and 50% Hydroxylamine (HA) were purchased from Thermo Fisher Scientific (Waltham, MA). Ethanol was purchased from Decon Labs, Inc. (King of Prussia, PA).
4
Affinity-based assembly of T4-Soc/Hoc-AAV vectors
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For assembly of T4-Soc-AAV vectors on nickel beads, T4-Soc(His6)-biotin vectors were loaded onto Ni2+-NTA (nitrilotriacetic acid) agarose beads (Qiagen, The Netherlands). After incubation for 1 hour at 4°C, the mixture was centrifuged at 100g for 30 s and washed five times with binding buffer. Avidin dissolved in binding buffer was then added to the beads. After 20 min of incubation at 4°C, free avidin was removed by washing and centrifugation. BioAAV vectors were then added to the T4-Soc-SBA immobilized beads and incubated for 30 min. After washing five times with binding buffer, bound T4-Soc-AAV vectors were eluted with elution buffer [50 mM tris-HCl (pH 8.0), 300 mM NaCl, and 300 mM imidazole]. The protocol for assembling T4-Hoc-AAV on nickel beads was similar to that used for T4-Soc-AAV. Last, the eluted vectors were exchanged into PBS-MK buffer.
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