Journal: Journal of visualized experiments : JoVE

Article Title: Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

doi: 10.3791/61243

Figure Lengend Snippet: Materials

Article Snippet: Ensure that the tube caps are tightened and the samples are balanced inside the homogenizer. table ft1 table-wrap mode="anchored" t5 caption a7 Name Company Catalog Nu2mber Comments 15 mL tube VWR 89039–664 STEP 1.2 2x Laemmli Sample Buffer BioRed 1610737 STEP 3.11 4–20% Mini-PROTEAN TGX Precast Protein Gels BioRed 4561096 STEP 4.1 anti-AIN-1 monoclonal antibody custom generated n/a STEP 4.2, see ref. Zhang et al. 2007 anti-ALG-1 monoclonal antibody custom generated by PRF&L n/a STEP 4.2 anti-HRPK-1 monoclonal antibody custom generated by PRF&L n/a STEP 4.2 Bullet Blender Storm Homogenizer MidSci BBY24M STEP 2.3 DL-Dithiothreitol (DTT) Sigma D9779–5G Dynabeads Protein A for Immunoprecipitation Thermo Fisher 10002D STEP 3.2 DynaMag-2 Magnet Thermo Fisher 12321D STEP 3.2 EDTA-free protease inhibitors Roche 11836170001 GFP antibody (FL) Santa Cruz Biotechnology sc-8334 Glycerol Thermo Fisher G33–500 Goat Anti-Rabbit Secondary Antibody, HRP BioRed 1662408 STEP 4.2 Goat anti-Rat IgG (H+L) Secondary Antibody, HRP Thermo Fisher 31470 STEP 4.2 HEPES Sigma H4034–500G LICOR WesternSure PREMIUM Chemiluminescent Substrate, 100 mL Kit LI-COR 926–95000 STEP 4.3 Magnesium chloride hexahydrate ACS VWR VWRV0288–500G Magnesium Sulfate Anhydrous Thermo Fisher M65–500 Microcentrifuge Tubes, 1.5 mL VWR 20170–333 STEP 1.6 N2 wild type CGC Navy RINO RNA Lysis Kit 50 pack (1.5 mL) MidSci NAVYR1-RNA STEP 2.3 Phosphatase inhibitor cocktail 2 Sigma P5726–1ML Phosphatase inhibitor cocktail 3 Sigma P0044–1ML Potassium Chloride Thermo Fisher P217–500 Potassium phosphate monobasic Thermo Fisher P285–3 RC DC Protein Assay Kit I BioRed 5000121 STEP 2.9 RNaseOUT Recombinant Ribonuclease Inhibitor Thermo Fisher 10777019 Sodium Chloride Thermo Fisher S271–500 Sodium Phosphate Dibasic Anhydrous Thermo Fisher S374–500 TritionX-100 Sigma X100–500ML UY38 hrpk-1(zen17) available upon request VT1367 col-19::gfp(maIS105) available upon request VT3841 alg-1(tm492) available upon request Open in a separate window Materials Homogenize the sample at the highest speed (setting 12) for 4 min.

Techniques: Generated, Immunoprecipitation, Lysis, DC Protein Assay, Recombinant