Pellets containing a mix of embryos, larvae and adults were prepared by collecting the animals (grown on NGM agar plates with E. coli OP50 bacteria) in M9 buffer and washing thrice before removing all of the buffer and snap-freezing in liquid nitrogen. Extracts were prepared by grinding the pellet with a mortar and pestle in the presence of liquid nitrogen and dissolving in Lysis Buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% Triton X-100, 5% glycerol (w/vol), 5 mg/ml cOmplete Proteinase Inhibitor Tablets (Roche, 11697498001), 1% P8340 (Sigma)). Extracts were cleared by centrifugation at 16100 x g for 5 minutes at 4°C. EEF-1A protein was enriched from the C. elegans extracts using a method previously employed for enrichment of eEF1A1 present in human cell extracts [21 (link)]. Extracts were loaded on Pierce Strong Cation Exchange (S) Spin Columns (Thermo Fisher Scientific, 90008) pre-equilibrated in Lysis Buffer, followed by extensive washing of the columns with Washing Buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5% glycerol (w/vol), 5 mg/ml cOmplete Proteinase Inhibitor Tablets (Roche, 11697498001), 1% P8340 (Sigma)). The retained material, including EEF-1A, was eluted from the columns with Elution Buffer (50 mM Tris-HCl pH 7.4, 400 mM NaCl, 5% glycerol (w/vol), 5 mg/ml cOmplete Proteinase Inhibitor Tablets (Roche, 11697498001), 1% P8340 (Sigma)).
Complete proteinase inhibitor tablets
Manufactured by Roche
Complete Proteinase Inhibitor Tablets are a laboratory reagent used to inhibit a broad range of proteolytic enzymes, including serine, cysteine, and metalloproteases. The tablets are designed to be added to cell or tissue lysates to prevent protein degradation during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pierce strong cation exchange s spin columns, by Thermo Fisher Scientific (1 mentions)
P8340, by Merck Group (1 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (1 mentions)
Anti actin antibody, by Cell Signaling Technology (1 mentions)
Nupage sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Mabe343, by Abcam (1 mentions)
Irdye 800cw conjugated goat anti rabbit secondary antibody, by LI COR (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
4 protocols using complete proteinase inhibitor tablets
1
Enrichment and Purification of EEF-1A Protein from C. elegans
2
Protein Extraction and Western Blotting
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Lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) supplemented with Complete proteinase inhibitor tablets (Roche) was used in homogenization of larvae. Western blotting was done according to manufacturer’s instructions and visualized by the Odyssey infrared imager (Li-Cor).
3
BRAF Protein Detection in Melanoma Cells
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Adherent melanoma cells were lysed in RIPA buffer (Thermo Scientific) supplemented with Complete Proteinase Inhibitor tablets (Roche) and quantified by bicinchoninic acid whole protein assay prior to SDS‐PAGE separation under reducing conditions. Separated proteins were transferred to a PVDF membrane via the iBlot dry blotting system (Life Technologies) and blocked in 10% cleared skimmed milk followed by detection with rabbit‐anti‐BRAF mAb clone 55C6 (Cell Signaling Technology). Equal protein loading was confirmed by membrane stripping and reprobing with anti‐Actin antibody (Cell Signaling Technology).
4
Protein Extraction and Analysis from Pellets
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Pellets containing a mix of embryos, larvae and adults were prepared as described above. Lysates were prepared by grinding the pellet with a mortar and pestle in the presence of liquid nitrogen and dissolving in lysis buffer (50 mM HEPES (pH 7.4), 150 mM KCl, 5 mM MgCl2, 0.1% Triton X-100, 5% glycerol (w/vol), 1 mM PMSF, 7 mg/ml cOmplete Proteinase Inhibitor Tablets (Roche, 11697498001). Lysates were cleared by centrifugation at 16100 x g for 20 minutes at 4°C. Protein concentrations were approximated by Bradford Assay (Bio-Rad). Samples were prepared by mixing 25 micrograms of protein extract with the required amount of 4x NuPAGE™ LDS Sample Buffer (Invitrogen, NP0007) and 10x NuPAGE Sample Reducing Agent (Invitrogen, NP0004), followed by heating at 70°C for 10 minutes. Proteins were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane by wet-transfer. The following primary antibodies were used: 1:1000 monoclonal mouse anti-eEF1A (Merck, 05–235), 1:10000 monoclonal mouse anti-puromycin (Merck, MABE343), 1:5000 polyclonal rabbit anti-actin (Abcam, ab8227). Detection was carried out with IRDye 680RD-conjugated goat anti-mouse secondary antibody (LI-COR Biosciences, 926–68070) or IRDye 800CW-conjugated goat anti-rabbit secondary antibody (LI-COR Biosciences, 926–32211) and infrared imaging (LI-COR Biosciences, Odyssey CLx).
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