Nupage 2 lds sample buffer

Whole cell lysates or subcellular fractions from 0.3–1 × 10⁷ cells, were immunoprecipitated with anti-FECH (S. Cruz, sc-377377ac), anti-RNAP1 (S. Cruz, sc-48385ac) or anti-CSB (A301-345A Bethyl) antibodies conjugated with A/G agarose beads according to standard protocols (54 (link)). Briefly, extracts were incubated overnight at 4°C with 15 μg of antibody-conjugated beads. Beads were washed three times with 1X IP high buffer (Active Motif) supplemented with 1 mg/ml BSA and three times with 1× IP high buffer. Immunoprecipitated proteins were eluted for 10 min at 70°C with NuPAGE-LDS Sample Buffer 2× (Thermo Fischer Scientific), supplemented with 50 mM DTT and further investigated by immunoblotting.
For the two-step co-immunoprecipitation (TIP), chromatin enriched fractions were first immunoprecipitated with anti-FECH antibody (S. Cruz, sc-377377ac) following the conditions described above. The immunoprecipitated proteins were eluted three times at RT with Ferrochelatase (A-3) blocking peptide (S.Cruz, sc-377377 P) and subsequently immunoprecipitated with anti-RPA194 antibody (S. Cruz, sc-48385ac). Final elution was performed for 10 min at 70°C with NuPAGE-LDS Sample Buffer 2× (Thermo Fischer Scientific) supplemented with 50 mM DTT.

Cells were treated with compounds as indicated. After treatment, the medium was collected from cells and incubated with StrataClean Resin (Agilent Technologies, Santa Clara, CA; 400714) in accordance with manufacturer’s instructions. Briefly, 10 μL of StrataClean Resin was added to 1 mL supernatant and incubated on a rotating wheel for 1 hour at 4°C. The resin was pelleted, and supernatant was removed. The remaining resin was incubated in 50 μL NuPAGE 2× LDS sample buffer (Thermo Scientific, NP0007) containing 5% beta-2-mercaptoethanol at 70°C for 10 minutes. Samples were separated by sodium dodecyl sulfate–polyacrylamide gel, and DKK-1 protein was detected by immunoblotting using anti-DKK-1 antibody.

For immunoprecipitation, cells were lysed in an immunoprecipitation specific lysis buffer (40 mmol/L Hepes [pH 7.5], 150 mmol/L NaCl, 1 mmol/L Na₂EDTA, 10 mmol/L sodium pyrophosphate, 50 mmol/L glycerophosphate, 50 nmol/L sodium fluoride, 1 mmol/L Na₃VO₄, 1 mmol/L phenylmethylsulfonyl fluoride, 0.5% NP40, 1× Roche complete EDTA-free protease inhibitor cocktail [Sigma-Aldrich, 1183617001], 1× Roche PhosSTOP [Sigma-Aldrich, 4906845001]) for 45 minutes on ice before clarifying at 13,000 rpm for 20 minutes. Protein concentrations were determined as previously described. For each reaction 5 μL of anti-E-cadherin (BD Transduction Laboratories, 610181) or anti-β-catenin (BD Transduction Laboratories, 610153) antibody was added to 800 μg protein and incubated at 4°C for 24 hours. Dynabeads protein G (Thermo Scientific, 10003D) were equilibrated in immunoprecipitation lysis buffer, and then 50 μL was added to each reaction and incubated for 16 hours at 4°C. Beads were washed in PBS containing 0.02% Tween20 three times before elution by incubating beads in 100 μL NuPAGE 2× LDS sample buffer (Thermo Scientific, NP0007) containing 5% beta-2-mercaptoethanol at 70°C for 10 minutes. Samples were separated by sodium dodecyl sulfate–polyacrylamide gel as previously described.