atg11Δ or SKY004 cells were transformed with the appropriate vectors. Cells were grown to mid-log phase in SMD. Protein expression was then induced with 0.5 μM copper (Sigma). After 3 hrs of induction, cells were pelleted (2,000 x g, 5 min) and either stored at −80°C for SMD conditions or resuspended in SD-N with 1 mM PMSF (Amresco) for 30 min and then harvested and stored at −80°C. Cells were resuspended in lysis buffer containing 1X phosphate-buffered saline (Corning), 0.2 M sorbitol (Sigma), 1 mM MgCl2 (VWR), 1% Triton X-100 (Alfa Aesar), 1 mM PMSF, and 1 complete EDTA-free tablet (Sigma Aldrich). Cells were lysed by bead beating and centrifuged at 16,100 x g at 4°C. Supernatant was incubated with FLAG M2 magnetic beads (Sigma), rocking for 2 hrs at 4°C. After removal of the flow-through, beads were washed 5 times with lysis buffer. 1 X NuPAGE LDS sample buffer (Invitrogen) was added to each tube. Tubes were then incubated at 37°C for 30 min to elute proteins bound to the magnetic beads. Supernatant was analyzed by western blot and probed with anti-FLAG (Sigma), anti-HA (Abcam) or anti-GFP (Santa Cruz) antibodies.
Complete edta free tablet
Manufactured by Merck Group
The Complete EDTA-free tablet is a laboratory reagent that provides a simple and effective solution for chelating metal ions without the use of EDTA. It is designed to facilitate various analytical and experimental procedures in a research setting.
Automatically generated - may contain errors
Lab products found in correlation
Phenyl methyl sulfonyl fluoride (pmsf), by Avantor (1 mentions)
Anti ha, by Abcam (1 mentions)
Mgcl2, by Avantor (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Flag m2 magnetic beads, by Merck Group (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Anti gfp, by Santa Cruz Biotechnology (1 mentions)
Anti flag, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Corning (1 mentions)
Bicinchoninic acid assay kit, by Thermo Fisher Scientific (1 mentions)
Protease phosphatase inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Brij 98, by Merck Group (1 mentions)
Brij 98, by Thermo Fisher Scientific (1 mentions)
3 protocols using complete edta free tablet
1
Affinity Purification of Protein Complexes
2
Quantitative Immunoprecipitation of NaV1.7 Channels
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Samples were isolated and lysed in 1% Triton-X-100, 25mM Hepes, 150 mM NaCl, 1 mM
EDTA buffer, 1 Complete EDTA-free Tablet (Sigma Aldrich, St. Louis, MO) using a
Covaris S2 Focused Ultrasonicator (Covaris, Woburn, MA). Protein concentration
was measured using bicinchoninic acid assay kit (ThermoFisher Scientific,
Waltham MA). NaV1.7 was immunoprecipitated with 10 µg
NaV1.7 Ab (Neuromab, Cat no. 75–103, the University of California
(UC) Davis/National Institutes of Health (NIH) NeuroMab Facility, Davis, CA) or
(Millipore Sigma Cat no. AB5390, Burlington, MA) or (Pan-Nav Ab, Alomone Labs,
Cat no. ASC-003, Jerusalem, Israel) overnight at 4°C, followed by PeggySue
(ProteinSimple, San Jose, CA) blotting with anti-NaV1.7 Ab (Neuromab,
Cat no. 75–103, UC Davis/NIH NeuroMab Facility, Davis, CA) followed by
Streptavidin (SA)-horse radish peroxidase (HRP) anti-mouse IgG H + L
(ProteinSimple, San Jose, CA) detection. Nav stable cells lines were as
previously described.26 (link)
EDTA buffer, 1 Complete EDTA-free Tablet (Sigma Aldrich, St. Louis, MO) using a
Covaris S2 Focused Ultrasonicator (Covaris, Woburn, MA). Protein concentration
was measured using bicinchoninic acid assay kit (ThermoFisher Scientific,
Waltham MA). NaV1.7 was immunoprecipitated with 10 µg
NaV1.7 Ab (Neuromab, Cat no. 75–103, the University of California
(UC) Davis/National Institutes of Health (NIH) NeuroMab Facility, Davis, CA) or
(Millipore Sigma Cat no. AB5390, Burlington, MA) or (Pan-Nav Ab, Alomone Labs,
Cat no. ASC-003, Jerusalem, Israel) overnight at 4°C, followed by PeggySue
(ProteinSimple, San Jose, CA) blotting with anti-NaV1.7 Ab (Neuromab,
Cat no. 75–103, UC Davis/NIH NeuroMab Facility, Davis, CA) followed by
Streptavidin (SA)-horse radish peroxidase (HRP) anti-mouse IgG H + L
(ProteinSimple, San Jose, CA) detection. Nav stable cells lines were as
previously described.26 (link)
3
Quantification of Hepatic Cytochrome P450s
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PB was purchased from Mallinckrodt (St. Louis, MO). Brij 98 was purchased from Acros Organics (distributed by Thermo Fisher (Fair Lawn, NJ)). The 100 × protease/phosphatase inhibitor cocktail used in the preparation of microsomes was purchased from Cell Signaling Technology (Danvers, MA). The protease inhibitor used in the Brij 98-mediated solubilization of microsomes was the "Complete" EDTA-free tablet from Sigma (St. Louis, MO). The primary antibodies used were as follows: anti-CYP1A1 from Everest Biotech (Oxfordshire, UK) (EB11171) used at 1:500; both anti-CYP3A (PM40) and anti-CYP2E1 (PA26) from Oxford Biomedical (Rochester Hills, MI) used at 1:1000 and 1:4000, respectively; anti-CYP2D6 (MBS821765) from MyBioSource (San Diego, CA) used at 1:1000. Protein standards used for westerns were purified rabbit CYP1A2 (Backes et al., 1998) ; purified, recombinant rabbit CYP2E1 (Cheng et al., 2004) ; purified N-truncated, recombinant CYP3A4 (Moutinho et al., 2012) ; and CYP2D6 human supersomes (Gentest-Corning). All other chemicals of reagent quality that were used were purchased from Sigma.
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