Cells grown to approximately 60% confluency were transfected with plasmids encoding Tau and the human kinase library generated in this study using Lipofectamine 2000 Reagent (Life Technologies) and Opti-MEM (Gibco). The cell culture medium was replaced the next day, and after a 48 h incubation period to allow for protein expression, the cells were trypsinized and collected by centrifugation. Cells were washed with ice-cold PBS once, then lysed in 0.4 ml of lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% NP-40) supplemented with protease inhibitor cocktail (Pierce) by sonication. After centrifugation of the lysate at the maximal speed for 30 min at 4 °C, the lysate supernatant was recovered, diluted to 1 ml with wash buffer (50 mM Tris–Cl [pH 7.5] and 150 mM NaCl) supplemented with protease inhibitor cocktail, and subjected to immunoprecipitation [IP] with GFP-Trap Magnetic Agarose beads (ChromoTek) for 1 h at 4 °C. Bead-bounded protein complexes were washed once with lysis buffer, once with radioimmunoprecipitation assay buffer (50 mM Tris–Cl [pH 7.5]; 150 mM NaCl; 0.5 mM EDTA; 0.1% SDS; 1% Triton X-100; and 1% deoxycholate), and once with wash buffer.
Gfp trap magnetic agarose beads
Manufactured by Proteintech
Sourced in Germany
GFP-Trap magnetic agarose beads are a laboratory product designed for the isolation and purification of GFP-tagged proteins. The beads are made of agarose and are coupled with a highly specific single-domain antibody fragment that binds to GFP. The magnetic properties of the beads allow for easy separation and washing of the captured proteins using a magnetic separator.
Automatically generated - may contain errors
Lab products found in correlation
Rnase inhibitor, by Thermo Fisher Scientific (5 mentions)
Protease inhibitor cocktail without edta, by Promega (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Benzonase nuclease, by Merck Group (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Complete edta free protease inhibitor cocktail, by Merck Group (2 mentions)
Hiseq 4000, by Illumina (2 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Nacalai Tesque (2 mentions)
Dynamag 2 magnet, by Thermo Fisher Scientific (2 mentions)
Anti α tubulin, by Merck Group (2 mentions)
Goat anti mouse hrp, by Jackson ImmunoResearch (2 mentions)
Olaparib, by Selleck Chemicals (2 mentions)
Goat anti rabbit hrp, by Jackson ImmunoResearch (2 mentions)
Anti flag m2 magnetic bead, by Merck Group (2 mentions)
Benzonase, by Merck Group (2 mentions)
Edta free protease inhibitor tablet, by Roche (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
72 protocols using gfp trap magnetic agarose beads
1
Tau Interactome Profiling Using Kinase Library
2
Protein Interaction Analysis of ACBD5 and VAPB
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For IP experiments GFP-tagged ACBD5 and Myc-tagged VAPB were expressed in COS-7 cells. After 48 h, cells were washed in PBS and then lysed in ice-cold lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM PMSF, and protease inhibitor cocktail), undissolved material was pelleted by centrifugation at 15,000 g, followed by a second centrifugation step at 100,000 g. Clarified lysates were then mixed with GFP-TRAP magnetic agarose beads (ChromoTek) and incubated for 2 h at 4°C. Beads were subsequently washed extensively with lysis buffer and bound proteins eluted with Laemmli buffer. Immunoprecipitates and total lysates were analyzed by Western immunoblotting. For in vitro binding assays, GST-VAPBΔTMD and MBP-ACBD5ΔTMD constructs were expressed in BL21 Rosetta (DE3) cells (EMD Millipore) induced with 1 mM IPTG for 4 h. Cells were pelleted by centrifugation at 5,000 g for 10 min and cell pellets resuspended in ice-cold E. coli lysis buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 0.1% NP-40, 1 mM PMSF, and protease inhibitor cocktail). Cells were disrupted by sonication, and the 15,000 g supernatant was incubated with GST-TRAP agarose beads (ChromoTek) for 2 h at 4°C. Beads were then washed extensively with E. coli lysis buffer and bound proteins eluted using Laemmli buffer. Immunoprecipitates and total lysates were subsequently analyzed by Western immunoblotting.
3
Immunoprecipitation of ORC2-GFP and ORR
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Immunoprecipitations were performed on three biological replicates of both ORC2-GFP and ORR embryos. For each replicate, 0.5 g of embryos aged 18–24 h were collected, dechorionated, and flash frozen. Frozen embryos were ground thoroughly with a mortar and pestle in liquid N2. Ground embryos were then resuspended in NP40 Lysis Buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl, 1% NP40, 1 mM EDTA, 1 mM EGTA, with 2X cOmplete™ Protease Inhibitor Cocktail EDTA-free (Millipore Sigma)). The embryonic extract was treated with benzonase nuclease (Millipore #7066) at a final concentration of 30 U/mL for 30 min at 4°C. After benzonase treatment, the extract was centrifuged at 4,000 rcf for 5 min. The supernatant was then used for the immunoprecipitation.
Prior to the immunoprecipitation, GFP Trap magnetic agarose beads (Chromotek #gtma-10) were washed and equilibrated with NP40 lysis buffer. Beads were added to extract and incubated for 2 h at 4°C. After the 2 h, beads were isolated and washed with 4 times with NP40 lysis buffer. Beads were then resuspended in 2× Laemmli sample buffer (Biorad #1610737) and boiled at 95°C for 20 min to elute protein.
Prior to the immunoprecipitation, GFP Trap magnetic agarose beads (Chromotek #gtma-10) were washed and equilibrated with NP40 lysis buffer. Beads were added to extract and incubated for 2 h at 4°C. After the 2 h, beads were isolated and washed with 4 times with NP40 lysis buffer. Beads were then resuspended in 2× Laemmli sample buffer (Biorad #1610737) and boiled at 95°C for 20 min to elute protein.
4
Immunoprecipitation of Transfected Proteins
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Transfected cells were washed with ice-cold PBS and lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 1% Triton X-100 [Sigma-Aldrich, T9284], 3% glycerol [Sigma-Aldrich, G6279]) with a protease inhibitor cocktail (Roche, 11697498001). Cell lysates were clarified by centrifugation at 13,000 g for 20 min and incubated with GFP-trap magnetic agarose beads (ChromoTek, gtma-20) or anti-MYC/c-myc agarose (Thermo Fisher Scientific, 20169) at 4°C for 4 h. After 3 washes with lysis buffer, the precipitates were eluted with 1X LDS loading buffer for GFP-trap precipitation or 3 × MYC peptide (Thermo Fisher Scientific, 20170) for anti-MYC precipitation. The precipitated samples were analyzed by immunoblotting with the indicated antibodies.
5
Worm RIP-seq Protocol for Transcriptome
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A total of ~100,000 synchronized young adult animals were used for RIP-seq. Worm pellets were resuspended in equal volumes of immunoprecipitation buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2.5 mM MgCl2, 0.5% NP-40, 80 U/mL RNase Inhibitor (Thermo Fisher Scientific), 1 mM dithiothreitol, and protease inhibitor cocktail without EDTA (Promega)), and grinded in a glass grinder for 8–10 times. Lysates were clarified by spinning down at 15,000 rpm, 4 °C, for 15 min. Supernatants were incubated with the GFP-Trap magnetic agarose beads (ChromoTek) at 4 °C for 1 h. Beads were washed with wash buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2.5 mM MgCl2, 0.5% NP-40, and 1 mM dithiothreitol) six times, and then resuspended in TBS buffer for RNA extraction. Total RNA was extracted using the standard method with TRIzol reagent (Invitrogen). Small RNA libraries for RNA-seq were prepared as described above. Libraries were sequenced using an Illumina HiSeq4000 to obtain single-end 36-nt sequences at the University of Chicago Genomic Facility.
6
GFP-Trap Immunoprecipitation of MyoE and Variants
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Cell extracts [strains, MyoE-GFP (MAD4406), UDS1-GFP (MAD6379), HMSV-GFP (MAD7326) and Uso1-GFP (MAD6358)] were prepared as described above, but using the lysis buffer recommended by the manufacturer, which containing 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP40 and cOmplete protease inhibitors. Approximately 100 mg of total protein (4 mL of extract) were immunoprecipitated with 25 μL of GFP-Trap magnetic agarose beads (Chromotek #gtma-20) following incubation for 2 h at 4°C in a rotating wheel. Beads were washed four times with the same buffer before eluting the immunoprecipitated material with 60 μL of Laemmli buffer. 10 μL aliquots were analyzed by α-HA3 western blotting (7.5% polyacrylamide gels) or α-MyoE western blotting. 2 μL were analyzed by α-GFP western blotting to determine levels of immunoprecipitated baits. Lastly 8 μL were analyzed by SDS-PAGE and silver staining. GFP-trap co-immunoprecipitation experiments of HMSV-HA3 with GFP-MyoE [GTD] and MyoE [ΔGTD] were carried out with otherwise myoEΔ strains MAD7864 and MAD7862, respectively.
7
GFP-Trap Affinity Purification Protocol
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Worms were prepared as described for the mRNA pulldown and lysed in lysis buffer (25 mM HEPES-KOH pH 7.4, 100 mM KoAC, 10 mM MglCl2, 0.5 mM DTT, 2 tablets EDTA free protease inhibitor) using a Biorupter (Diagenode). The lysate was clarified by centrifugation at 2000 x g for 10 min at 4oC. For each sample, 50 μl of GFP-Trap Magnetic Agarose beads (Chromotek) were prepared by washing the beads twice in 1 ml lysis buffer. Worm lysate (50 mg protein total) was adjusted to 10 mg/ml and added to the equilibrated beads. The suspension was then incubated for 2 h at 4oC with vertical rotation. The beads were washed once with 2 ml of lysis buffer and twice with 1 ml lysis buffer containing 0.1% NP-40. After transferring the beads to a fresh 1.5 ml tube, the beads were washed 3 times with lysis buffer without detergent. The bound protein was digested on beads using the iST 8x Kit (PreOmics) following manufacturer′s instructions.
8
GFP-Trap Magnetic Agarose Beads Enrichment
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HEK-293T cells were harvested ~24 hours after transfection and washed twice in ice-cold PBS. Cells were lysed in lysis buffer [100 mM tris-HCl, 150 mM NaCl (pH 7.4), 0.1% SDS, and 1% Triton X-100] supplemented with 1:100 Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific) with end-over-end rotation at 4°C for 1 hour. Lysates were cleared by centrifugation at 10,000g for 10 min, after which the supernatant was transferred onto equilibrated GFP-Trap Magnetic Agarose Beads (ChromoTek) with end-over-end rotation at 4°C for 2 hours. The magnetic beads were washed four times with lysis buffer before incubation in 2× SDS sample buffer (supplemented with 100 mM dithiothreitol) at 60°C for 20 min to elute bound proteins. Samples from cleared total lysates, supernatant (after incubation with magnetic beads), wash, and elution steps were detected with the rabbit anti-FLAG (701629; Invitrogen) antibody using the Western blot technique (described in detail above).
9
Immunoprecipitation from HEK293 Cells
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For sample preparation for immunoprecipitations (IP), HEK293 cells were grown on 10 cm dish and washed twice in phosphate-buffered saline (PBS: Nacalai Tesque, Inc.), and lysed in lysis buffer (100 mM NaCl, 0.2% NP-40, 1 mM MgCl2, 10% glycerol, 50 mM Tris-HCl, pH 7.5), supplemented with protease and phosphatase inhibitor cocktail. After 30 min-incubation on the rotator at 4°C, lysates were cleared by the centrifugation 20,000 x g for 20 mins at 4°C. Next, lysates were incubated with 10 μl of GFP-Trap magnetic agarose beads (ChromoTek, GmbH) for 2 h with mixing on a rotator at 4°C. Lysates were then washed five times with lysis buffer, and eluted in 2X SDS sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.01% bromophenol blue, 5% 2-mercaptoethanol).
10
ChIP-qPCR Analysis of Rice Chromatin
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ChIP assay was performed as described previously (Yamaguchi et al., 2014 (link)) with minor modifications. Briefly, a total of 1 g rice tissues were cross‐linked with 1% formaldehyde for 30 min. The cells were lysed and the nuclei‐containing fractions were isolated. The chromatins were sheared using a sonicator (Bioruptor, UCD‐300) to the size range of 200–1000 bp; 2% volume of the supernatants was recovered as input. The chromatin suspensions were immunoprecipitated with protein G magnetic beads (Invitrogen, 78608) conjugated to anti‐Flag antibody (MBL, M185‐3, Kyoto, Japan), or GFP‐Trap Magnetic Agarose beads (Chromotek, gtma‐20, Planegg‐Martinsried, Germany), and the WT and 35S:GFP transgenic rice plants were as negative controls. The precipitated DNA fragments were recovered and quantified by real‐time PCR analyses as described above. The % input of the target genes were determined by the cycle threshold in IP sample relative to that of the input sample using the formula 2^‐[cq IP‐(cqinput‐5.64)]. Relative enrichment of a gene was normalized to the respective negative controls. Primers and probes used in ChIP‐qPCR are listed in Table S6 .
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