C2C12 cells were transfected with Myc-tagged Ddx3x (NM_010028 .3), Ddx5 (NM_007840 .3), DGCR8 (NM_001190326 .1), Ezh2 (NM_007971 .2), Fus (NM_139149 .2), Fxr1 (NM_008053 .2), Stau1 (NM_017453 .3), Chronos 98-3259, Chronos 1467-3259, or pcDNA3.1+ using Lipofectamine 2000 (Thermo Fisher Scientific). Chronos 98-3259 and Chronos 1467-3259 were cloned into the pcDNA3.1+ vector. Cells were lysed in 10 mM Hepes, pH 7.9; 100 mM KCL; 2 mM MgCl2; 0.2 mM DTT; 200 U/ml RNase inhibitor; 1 mM ATP; 0.2 mM GTP; and 0.5% Triton X-100 and cleared by centrifugation at 10,000 g for 10 min at 4°C. Cleared lysates were diluted 1:3 with lysis buffer and incubated with EZview RED anti-c-Myc affinity gel (Sigma-Aldrich) at 4°C for 6 h with gentle rocking. Beads were subsequently washed five times with lysis buffer. C0mplete EDTA-free protease inhibitor (Roche) was present in all solutions from initial lysis to final wash. For protein analysis, beads were resuspended in 50 µl of 2× Laemmli buffer and stored at −20°C until size separation by SDS-PAGE and Western blot analysis. For RNA analysis, beads were resuspended in 1 ml of TRIzol (Thermo Fisher Scientific), and RNA was purified according to manufacturer’s protocol. One-half of the RNA was reverse transcribed using random primers, and the other half was reserved as a negative control.
Ezview red anti c myc affinity gel
Manufactured by Merck Group
EZview red anti-c-myc affinity gel is a chromatography resin used for the purification of c-myc-tagged proteins. It is made up of agarose beads conjugated with an anti-c-myc antibody, which specifically binds to proteins containing the c-myc epitope tag.
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Lab products found in correlation
Ezview red anti ha affinity gel, by Merck Group (4 mentions)
Protease inhibitor mix, by Roche (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Edta free protease inhibitor, by Roche (1 mentions)
Mouse igg agarose, by Merck Group (1 mentions)
Proteinase inhibitor cocktail, by Merck Group (1 mentions)
Anti mouse igg conjugated to alkaline phosphatase, by Merck Group (1 mentions)
Radio immunoprecipitation assay buffer ripa buffer, by Thermo Fisher Scientific (1 mentions)
Vectastain elite abc, by Vector Laboratories (1 mentions)
Glutathione sepharose resin, by BD (1 mentions)
Flag peptide, by Merck Group (1 mentions)
Flag m2 agarose beads, by Merck Group (1 mentions)
Anti myc, by Roche (1 mentions)
Anti ha, by Roche (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti ha affinity matrix, by Roche (1 mentions)
Anti ha antibody, by Merck Group (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
23 protocols using ezview red anti c myc affinity gel
1
RNA-binding protein interactome in C2C12 cells
2
Affinity Purification of Epitope-Tagged Mitochondrial Proteins
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Digitonin-extracted mitochondria-enriched fractions of 1 × 108 cells expressing the epitope-tagged protein of interest were solubilized for 15 min on ice in 20 mM Tris-HCl pH7.4, 0.1 mM EDTA, 100 mM NaCl, 10% glycerol containing 1X Protease Inhibitor mix (Roche, EDTA-free) and 1% (w/v) digitonin. After centrifugation (15 min, 20’000 g, 4°C), the lysate (input) was transferred to either 50 μl of HA bead slurry (anti-HA affinity matrix, Roche) or 30 μl c-myc bead slurry (EZview red anti-c-myc affinity gel, Sigma) both of which had been equilibrated in wash buffer (20 mM Tris-HCl pH 7.4, 0.1 mM EDTA, 100 mM NaCl, 10% glycerol, 0.2% (w/v) digitonin). After incubating at 4°C for 1 hr, the supernatant containing the unbound proteins was removed. The bead slurry was washed three times with wash buffer and the bound proteins were eluted by boiling the resin for 5 min in 2% SDS in 60 mM Tris-HCl pH 6.8 (IP). Five percent of both the input and the unbound proteins and 100% of the IP sample were subjected to SDS-PAGE and Western blotting.
For the stalled import intermediate, LDH-DHFR-HA expression was induced by tetracycline and respective cell cultures were supplemented with 1 mM sulfanilamide and 50 μM aminopterine (AMT) 12 hr before the experiment (Harsman et al., 2016 (link)). CoIP was performed as described above.
For the stalled import intermediate, LDH-DHFR-HA expression was induced by tetracycline and respective cell cultures were supplemented with 1 mM sulfanilamide and 50 μM aminopterine (AMT) 12 hr before the experiment (Harsman et al., 2016 (link)). CoIP was performed as described above.
3
Affinity Purification of Protein Complexes
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Digitonin-extracted mitochondria-enriched fractions of 1 × 108 induced cells were solubilized on ice in 20 mM Tris–HCl (pH 7.4), 0.1 mM EDTA, 100 mM NaCl, 25 mM KCl, 1x protease inhibitor mix (EDTA-free; Roche), and 1% (wt/vol) digitonin. After centrifugation (15 min, 20817g, 4°C), the lysate (IN, input) was transferred to either 50 μl of HA bead slurry (anti-HA affinity matrix; Roche) or 50 μl c-myc bead slurry (EZview red anti-c-myc affinity gel; Sigma-Aldrich), both of which had been equilibrated in wash buffer (20 mM Tris–HCl [pH 7.4], 0.1 mM EDTA, 100 mM NaCl, 10% glycerol, 0.2% [wt/vol] digitonin). After incubating at 4°C for 2 h on a rotating wheel, the supernatant containing the unbound proteins (FT, flow through) was removed. The bead slurry was washed three times with wash buffer. Bound proteins were eluted by boiling the resin in 60 mM Tris–HCl (pH 6.8) containing 2% SDS (IP). 2.5% of crude mitochondrial fractions (Input, IN), unbound proteins in the flow through (FT), and 50% of the final eluates (IP) were separated by SDS–PAGE and analysed by Western blot.
4
Immunoprecipitation of c-Myc and HA
5
Affinity Purification of GST- and c-myc-Tagged Proteins
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The plasmids encoding the N-terminal Glutathione S-transferase (GST) or c-myc tag linked to the proteins being investigated are listed in Supplementary Table S1 . All GST- and c-myc-tagged proteins were expressed in E. coli Rosetta (DE3). Total proteins containing c-myc fusion proteins (EBF1 or EBF2) were extracted from bacteria by sonication in Radioimmunoprecipitation assay buffer (RIPA buffer;Thermo Fisher Scientific) supplemented with Proteinase Inhibitor Cocktail (Sigma-Aldrich) and purified on EZview™ Red Anti-c-myc Affinity Gel (Sigma-Aldrich) according to the manufacturer’s protocol. Next, the crude extracts containing GST fusion proteins [SLIM1_C1 (aa: 287–428), SLIM1_C2 (aa: 418–568) or SLIM1_Nt (aa: 1–286)] were incubated with above-prepared raisins at 4°C overnight with gentle rocking. The raisins were washed six times with RIPA buffer, boiled with 2× SDS–PAGE gel loading buffer, subjected to SDS–PAGE and next immunoblotted using mouse monoclonal anti-GST IgG and anti-c-myc IgG (Sigma-Aldrich, G1160 and Santa Cruz Biotechnology Inc., SC-40) as primary antibody and anti-mouse IgG conjugated to alkaline phosphatase (Sigma-Aldrich, A3562) as a secondary antibody.
6
Biotinylation and Immunoprecipitation of c-Myc
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Transfected HEK293 cells were washed with PBS and incubated with EZ-link Sulfo-NHS-LC-Biotin (21335, Pierce, Nepean, Ontario) diluted to 0.5 mg/mL in PBS for 30 min at 4°C. Cells were washed with PBS containing 100 mmol/L glycine and lysed in RIPA buffer as described above. Supernatant was incubated overnight at 4°C with EZview Red Anti-c-Myc Affinity gel (E6654, Sigma Aldrich) and eluted with Laemmli buffer containing 2% (v/v) β-mercaptoethanol. Blotting proceeded as described above; however, the membrane was blocked with TBS-T containing 5% (w/v) bovine serum albumin (BSA) and incubated with Vectastain ABC Elite as per manufacturer’s instructions (PK-6100, Vector Laboratories, Burlingame, CA) before being exposed to film.
7
HIGD-1B Protein Interaction Assay
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Recombinant GST-fused HIGD-1B protein was expressed and purified from Escherichia coli BL21 (DE3). The purification of OPA1-Myc was performed using a EZview red anti-c-Myc affinity gel (Sigma-Aldrich; Merck KGaA) according to the manufacturer's instructions. GST pull-down assays were performed by incubating HIGD-1B-GST, immobilized on glutathione-sepharose resin (BD Biosciences) with OPA1-Myc at 4°C for 3 h. The beads were washed three times to remove unbound protein, and the bound proteins were eluted, separated using SDS-PAGE and detected using western blot analysis according to standard western blotting procedures as described above.
8
Calpain-mediated RhoA cleavage analysis
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Lysates of COS-7 cells transiently transfected with either 2Xmyc WT-RhoA or Flag-RhoA 1–56 were collected in calpain digestion buffer (50 mM Tris-HCl pH 7.4, 100 mM KCl, 1 mM EDTA, 5 mM DTT) without protease inhibitors. Immunoprecipitation was performed with either Flag M2 agarose beads (Sigma-Aldrich, St-Louis, MO) or EZview red anti-c-myc affinity gel (Sigma-Aldrich, St-Louis, MO). Flag-tagged proteins were eluted with 5 μg ml-1 Flag peptide (Sigma-Aldrich, St-Louis, MO) in calpain digestion buffer. For in vitro digestion, 9 units of recombinant μ-calpain from human erythrocytes (EMD Millipore/Calbiochem, La Jolla, CA) were added to the purified lysates for 45 min at room temperature in the presence of 2 mM CaCl2 and the reaction was stopped by the addition of Laemmli sample buffer. As a negative control, 14 μM calpeptin (EMD Millipore/Calbiochem, La Jolla, CA) was added to the samples to inhibit calpain activity.
9
Co-immunoprecipitation of Shh and Kif5B Proteins
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Proteins of interest were expressed in NIH3T3 or HEK293T cells. Cell lysates were prepared ~48 hr post-transfection using RIPA lysis buffer (Millipore, Burlington, MA). Co-immunoprecipitation assays were performed as described (Marada et al., 2015 (link)) with the following modifications. EZview Red Anti-HA Affinity Gel (Sigma) and EZview Red Anti-c-Myc Affinity Gel (Sigma) were used to immunoprecipitate HA and Myc epitope-tagged proteins respectively. Immunoprecipitates were analyzed by western blot using the following antibodies: Anti-HA (1:2000, Roche), anti-Shh (1:2000, SCBT), anti-Kin/mKif5B (1:10,000, Cell Signaling), anti-Myc (1:1000, Roche), anti-Flag (1:2000, Sigma), anti-Furin (1:1000, SCBT) and anti-Tub (1:10,000, Cell Signaling).
10
Co-immunoprecipitation of Tagged Mitochondrial Proteins
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For co-immunoprecipitations of tagged TbTim11, TbTim12 and TbTim13, digitonin-extracted crude mitochondrial fractions or submitochondrial fractions of 1∙108 cells each were solubilized for 15 min at 4°C in 20 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 100 mM NaCl, 10% glycerol containing 1% (w/v) digitonin and 1X Protease Inhibitor mix (EDTA-free, Roche). Following a clearing spin (20,000 g, 15 min, 4°C), the lysate (load) was transferred to affinity purification resin (30 μl EZview red anti-c-myc affinity gel from Sigma or 50 μl anti-HA affinity matrix from Roche) that had been equilibrated in wash buffer (20 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 100 mM NaCl, 10% glycerol containing 0.2% (w/v) digitonin). After 2 h of incubation at 4°C, the supernatant (unbound proteins) was removed and the resin was washed 3 times with 500 μl wash buffer. To elute the bound proteins, the resin was boiled for 5 min in 2% SDS in 60 mM Tris-HCl pH 6.8 (eluate). The resulting samples were analyzed by SDS-PAGE and Western blotting.
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