For pull-down assay, HEK293T cells were transfected with eGFP constructs using Lipofectamine LTX (Thermo Fisher). The following day, cells were washed and lysed in PBS with 0.1% Triton X-100 and EDTA-free protease inhibitor cocktail (Nacalai Tesque), passed through a 27-gauge needle 7 times, incubated on ice for 30 min, and centrifuged at 20k x g for 20 min. eGFP fluorescence per mg total protein was calculated, and lysate from untransfected cells was used to adjust for equivalent eGFP fluorescence and protein concentration between different eGFP lysates. Pull-down samples were prepared by incubating 1 mg/mL total protein concentration with equivalent eGFP fluorescence and 1 mM GST protein for 4 hr at 4 C with end-over-end rotation. Glutathione Sepharose 4B beads (GE Healthcare) were then added and further incubated for 1 hr. Samples were centrifuged at 500x g, and beads were washed four times in PBS supplemented with an additional 150 mM NaCl, 0.1% Triton X-100, and EDTA-free protease inhibitor cocktail (Nacalai Tesque). Beads were eluted in Laemmli buffer for 5 min at 95 C and separated by SDS-PAGE as described above.
Edta free protease inhibitor cocktail
Manufactured by Nacalai Tesque
Sourced in United States, Japan
EDTA-free protease inhibitor cocktail is a laboratory product that inhibits the activity of proteases, enzymes that break down proteins. This solution does not contain EDTA, a common chelating agent. The core function of this product is to preserve protein integrity during extraction and purification processes.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (1 mentions)
Prostain protein quantification kit, by Active Motif (1 mentions)
Las 4000 mini, by Fujifilm (1 mentions)
Horseradish peroxidase conjugated anti mouse or anti rabbit antibodies, by GE Healthcare (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor, by Nacalai Tesque (1 mentions)
Las 4000, by Cytiva (1 mentions)
Dynabeads antibody coupling kit, by Thermo Fisher Scientific (1 mentions)
Ab22552, by Abcam (1 mentions)
Bioruptor ucd 300, by Cosmo Bio (1 mentions)
Anti ha antibody beads, by Fujifilm (1 mentions)
Polytron pt1200 homogenizer, by Kinematica (1 mentions)
Chemi lumi one l kit, by Nacalai Tesque (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Protein a sepharose beads, by GE Healthcare (1 mentions)
Glycerol, by Thermo Fisher Scientific (1 mentions)
Anti flag m2 antibody, by Merck Group (1 mentions)
Las4000, by GE Healthcare (1 mentions)
16 protocols using edta free protease inhibitor cocktail
1
Protein-Protein Interaction Assay in HEK293T
2
Western Blotting of Smad2 and ERK Signaling
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Western blotting was performed using standard procedures as described [12 (link)]. Briefly, cells were seeded in adherent plates and cultured in the RPMI 1640 medium. Cells were starved overnight before treatment with 200 ng/mL of recombinant human GDF15 or 200 ng/mL of recombinant human TGF-β. SB431542 or U0126 was added 1 hour before treatment with GDF15 or TGF-β. Proteins were collected at the indicated times using RIPA buffer (Thermo Fisher Scientific) supplemented with a phosphatase inhibitor (Nacalai Tesque) and an EDTA-free protease inhibitor cocktail (Nacalai Tesque). Proteins were quantified using ProStain Protein Quantification Kit (Active Motif, Carlsbad, CA, cat. #15001). Anti-Smad2 (cat. #5339), p-Smad2 (cat. #3108), ERK1/2 (cat. #9122), and p-ERK1/2 (cat. #9101) antibodies were purchased from Cell Signaling Technology. Proteins were detected with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies (GE Healthcare, Little Chalfont, United Kingdom). The LAS 4000 mini (Fujifilm, Tokyo, Japan) was used to detect the blots.
3
Immunoprecipitation and detection of protein post-translational modifications
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Cells were lysed in IP buffer (20 mM Tris–HCl (pH 7.4), 200 mM NaCl, 2.5 mM MgCl2, 0.2% NP-40, 1 mM PMSF, 10 mM NAM, 1 μM TSA and EDTA-free protease inhibitor cocktail (Nacalai Tesque)), and were disrupted by sonication with two 30-s pulses at an interval of 30 s at 4 °C (Bioruptor UCD-300, CosmoBio). Calvariae harvested from 10-week-old male Sirt7 KO mice or WT mice were homogenized with a Polytron PT1200 homogenizer (Kinematica AG, Switzerland) in IP buffer at 4 °C. Soluble fractions were separated by centrifugation at 14,000×g for 10 min at 4 °C. Then the cell lysate was mixed with anti-HA antibody beads (Wako) or anti-OSX-conjugated magnetic beads, which were prepared using 10 μg of OSX antibody (ab22552, Abcam) and 1 mg of beads according to the protocol of the Dynabeads Antibody Coupling Kit (Invitrogen), and was stirred for 15 h at 4 °C. After washing with IP buffer (the same composition as above except for 1% NP-40), proteins were eluted with 1× SDS sample buffer (100 mM Tris–HCl pH 6.8, 4% SDS, 20% glycerol and 0.2% bromophenol blue). Finally, acetylation and propionylation of lysine were detected by western blotting with pan anti-Ac-K antibody (Cell Signaling Technology) and an anti-propionyllysine rabbit polyclonal antibody (PTM Biolabs Inc., LOT#Z338F112P3), respectively.
4
Affinity Purification of mCherry-IFT54
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IFT54-KO cells stably expressing an mCherry-fused IFT54 construct were grown to almost confluency on a 15-cm plate. The cells were lysed in 1 mL of lysis buffer (20 mM HEPES-KOH [pH 7.4], 3 mM MgCl2, 1 mM dithiothreitol, 100 mM KCl, 10% glycerol, 5 mM NaCl, and 0.1% Triton X-100) containing EDTA-free protease inhibitor cocktail (Nacalai Tesque) by placing on ice for 40 min. After centrifugation of the lysates at 16,000×g at 4°C for 20 min, the supernatants were incubated with 40 µl of GST-tagged anti-mCherry Nb prebound to glutathione-Sepharose 4B beads. The beads were washed five times with the lysis buffer and boiled in SDS-PAGE sample buffer. The proteins bound to the beads were then separated by SDS-PAGE, and electroblotted onto an Immobilon-P membrane (Merck Millipore). The membrane was blocked in 5% skimmed milk and incubated sequentially with the primary antibody and the peroxidase-conjugated secondary antibody. Protein bands were detected with a Chemi-Lumi one L kit, Chemi-Lumi super kit or Chemi-Lumi ultra kit (Nacalai Tesque).
5
FLAG-TARDBP Immunoprecipitation Assay
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The HBV producing cell line T23 was transfected with 5 μg FLAG-TARDBP for 48 hours. The cells were lysed in ice cold Pierce IP lysis buffer consisting of 25 mM Tris/HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol (Thermo Scientific) supplemented with EDTA-free protease inhibitor cocktail (Nacalai), and incubated on ice for 5 minutes with periodic mixing. The lysates were transferred to a micro-centrifuge tube and centrifuged at 13k × g for 10 minutes to pellet the cell debris at 4 °C. The supernatant was immunoprecipitated overnight with 5 μg anti-FLAG M2 antibody (Sigma) in the presence of protein A Sepharose beads (GE Healthcare). As a control, half of the lysate was immunoprecipitated with normal mouse IgG (MBL, Life science). The beads were washed four times in ice cold lysis buffer, treated with SDS sample buffer before being subjected to SDS-PAGE electrophoresis to detect proteins.
6
Testis Protein Extraction and Western Blot
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Testes lysates were prepared in a solution of 150 mM NaCl, 1% Nonidet P-40, 10 mM Tris–HCl (pH 8.0) containing EDTA–free protease inhibitor cocktail (Nacalai) and phenylmethylsulfonyl fluoride. The soluble materials were resolved on SDS–4–20% polyacrylamide gel (Bio-Rad Lab, Berkeley, CA, USA) and transferred onto a polyvinylidene difluoride membrane (Bio-Rad Lab). The filters were blocked with 5% skim milk in PBS containing 0.1% Tween20 and then sequentially incubated with primary and secondary antibodies. The blots were developed by Chemi–Lumi One Super (Nacalai) and signals were detected by LAS4000 (GE Healthcare, Chicago, IL, USA). For the detection of USP26, USP26 was immunoprecipitated from testes lysates with anti–USP26 antibody Ab2 (see Antibodies) and the precipitated proteins were subjected to Western blot analysis.
7
Cell Culture of K562 Leukemia Cells
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Reagents were purchased from Sigma-Aldrich, and cell culture media from Invitrogen unless otherwise stated. Phosphate buffered saline (PBS) and EDTA-free protease inhibitor cocktail was purchased from Nacalai Tesque. K562 cells were sourced from ATCC and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS, Biowest), 100 units/mL penicillin and streptomycin, non-essential amino acids, and 0.3 g/L L-glutamine. The cells were expanded to a maximum of 1 x 106 cells/mL.
8
Immunoprecipitation of S100A13 Protein
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For immunoprecipitation (i.p.p.t), protein G-Sepharose™ beads (GE Healthcare Bio-Science Corp, Piscataway, NJ, USA)-captured rabbit anti-S100A13 antibody were added to conditioned medium (CM) in the presence of ethylenediaminetetraacetic acid (EDTA) free protease inhibitor cocktail (Nacalai Tesque) and incubated on a rotor for 2 h at 4 °C. The beads were washed three times with medium without serum and quenched with 50 µL of SDS sample buffer.
9
Expression and Purification of Human HAT Protein
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Gene encoding human HAT protein (BC063003) was cloned into pSUMO-LIC vector and expressed in BL21 (DE3) Rosetta T1R E. coli (Novagen) in Terrific Broth media supplemented with kanamycin and chloramphenicol. Cells were cultured and induced with 0.5 mM isopropyl-beta-d -1-thiogalactopyranoside (IPTG) at 18 °C overnight, harvested and resuspended in lysis buffer (100 mM HEPES, 500 mM NaCl, 10 mM imidazole, 10% (v/v) glycerol at pH 8.0) supplemented with 1:1000 (v/v) EDTA-free protease inhibitor cocktail (Nacalai) and 250U/ml of Benzonase (Merck). After sonication, centrifugation and clarified by filtration, the protein extract was loaded onto Ni-NTA Superflow column (Qiagen), washed and eluted with 5 column volumes of elution buffer (20 mM HEPES, 500 mM NaCl, 500 mM imidazole, 10% (v/v) glycerol at pH 7.5). Eluate was then loaded onto a HiLoad 16/60 Superdex-200 column (GE Healthcare) and eluted with 1 column volumes of elution buffer (20 mM HEPES, 300 mM NaCl, 10% (v/v) glycerol at pH 7.5). Relevant protein fractions were pooled and concentrated using centrifugal force driven filter concentrators (VivaScience). Protein purity was assessed on SDS-PAGE and identity confirmed by mass spectrometry analysis. Protein concentration was determined by the absorbance at 280 nm using Nanodrop spectrophotometer (ThermoFisher Scientific).
10
Visualizing Protein-Protein Interactions
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The VIP assay and subsequent immunoblotting analysis were performed by a previously described method (Katoh et al., 2015 (link), 2016 (link)) with minor modifications (Nishijima et al., 2017 (link)). Briefly, approximately 1.6×106 HEK293T cells in six-well plates were transfected with EGFP and mChe fusion constructs using Polyethylenimine Max (20 µg, Polysciences, Warrington, USA), and cultured for 24 h. The transfected cells were then lysed for 20 min on ice in 250 µl of HMDEKN cell lysis buffer [10 mM HEPES (pH 7.4), 5 mM MgSO4, 1 mM DTT, 0.5 mM EDTA, 25 mM KCl, and 0.05% NP-40] containing EDTA-free protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan), and centrifuged at 16,100×g for 15 min at 4°C in a microcentrifuge. The supernatants (200 µl) were incubated with 5 µl of GST–anti-GFP Nb beads for 1 h at 4°C. After washing three times with 180 µl of the cell lysis buffer, the precipitated beads were observed using an all-in-one-type fluorescence microscope (BZ-8000, Keyence, Osaka, Japan) using a 20×/0.75 objective lens under constant conditions (sensitivity ISO 400, exposure 1/30 s for green fluorescence; and sensitivity ISO 800, exposure 1/10 s for red fluorescence).
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