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Protease complete

Manufactured by Merck Group
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Protease Complete is a laboratory product designed to facilitate the extraction and purification of proteins. It contains a blend of proteolytic enzymes that assist in the digestion and breakdown of protein samples. The product is intended for use in various research and analytical applications that require the isolation and preparation of proteins from complex mixtures.

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Lab products found in correlation

Mammalian protein extraction reagent, by Thermo Fisher Scientific (2 mentions) Restore plus buffer, by Thermo Fisher Scientific (2 mentions) Phosstop, by Merck Group (2 mentions) Chemidoc mp, by Bio-Rad (2 mentions) Laemmli sample buffer, by Bio-Rad (2 mentions) Krebs ringer buffer, by Merck Group (1 mentions)

Close spelling variants of this product

COmplete EDTA-free protease inhibitor cocktail COmplete ™ protease inhibitor complex COmplete EDTA-free protease inhibitor Complete mini protease inhibitor COmplete Mini Protease Inhibitor Cocktail Complete ULTRA protease inhibitor cocktail Complete protease and phosphatase inhibitor cocktails EDTA-free complete mini protease inhibitors COmplete Mini EDTA-free protease inhibitor COmplete mini protease inhibitor tablet

3 protocols using protease complete

1

Protein Extraction and Immunoblot Analysis

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Cells were lysed on ice for 10 minutes in freshly prepared Mammalian Protein Extraction Reagent (Thermo Scientific) supplemented with 1X Protease Complete, EDTA-free (Sigma) and 1X PhosSTOP (Sigma) before centrifugation at 15,000 rpm for 12-min at 4°C. Protein lysates were prepared using 4× Laemmli sample buffer (BioRad) heated in boiling water for 5-min. Immunoblot analysis was conducted with the antibodies listed in Supplementary Table S5. Immunoblot analysis of Akt phosphorylation was performed sequentially using the same PVDF membrane, first probing for phosphorylated Akt (Ser473), then total Akt. PVDF membranes were stripped for 8-min using Restore PLUS buffer (Thermo Scientific) and blocked with 5% BSA between subsequent probes. Tubulin was used a loading control for all immunoblot experiments. Images were digitally captured using a Bio-Rad ChemiDoc MP and analyzed with ImageJ.
Abreu D., Asada R., Revilla J.M., Lavagnino Z., Kries K., Piston D.W, & Urano F. (2020). Wolfram syndrome 1 gene regulates pathways maintaining beta cell health and survival. Laboratory investigation; a journal of technical methods and pathology, 100(6), 849-862.
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2

Erythrocyte Membrane Isolation and Radiolabeling

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Erythrocytes were washed and resuspended (50% haematocrit) with calcium-free Krebs-Ringer buffer (Sigma). 125 μL of cell suspension was incubated for 2 hr with 125 μCi of 32P (Perkin Elmer) and either 18 μg of EBA-175 RII or the equivalent volume of Krebs-Ringer buffer. Suspensions were spun down at 3000 g for 4 min at room temperature and the supernatant was discarded. Erythrocyte pellets were lysed with ice cold 5 mM sodium phosphate pH eight buffer with protease (cOmplete, Sigma) and phosphatase (Halt, Thermo Fisher) inhibitors, spun down at 100000 g and washed three times with the same buffer. The resulting ghost pellets were snap-frozen in liquid Nitrogen and stored at −80°C until further use.
Sisquella X., Nebl T., Thompson J.K., Whitehead L., Malpede B.M., Salinas N.D., Rogers K., Tolia N.H., Fleig A., O’Neill J., Tham W.H., David Horgen F, & Cowman A.F. (2017). Plasmodium falciparum ligand binding to erythrocytes induce alterations in deformability essential for invasion. eLife, 6, e21083.
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3

Protein Extraction and Immunoblot Analysis

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Cells were lysed on ice for 10 minutes in freshly prepared Mammalian Protein Extraction Reagent (Thermo Scientific) supplemented with 1X Protease Complete, EDTA-free (Sigma) and 1X PhosSTOP (Sigma) before centrifugation at 15,000 rpm for 12-min at 4°C. Protein lysates were prepared using 4× Laemmli sample buffer (BioRad) heated in boiling water for 5-min. Immunoblot analysis was conducted with the antibodies listed in Supplementary Table S5. Immunoblot analysis of Akt phosphorylation was performed sequentially using the same PVDF membrane, first probing for phosphorylated Akt (Ser473), then total Akt. PVDF membranes were stripped for 8-min using Restore PLUS buffer (Thermo Scientific) and blocked with 5% BSA between subsequent probes. Tubulin was used a loading control for all immunoblot experiments. Images were digitally captured using a Bio-Rad ChemiDoc MP and analyzed with ImageJ.
Abreu D., Asada R., Revilla J.M., Lavagnino Z., Kries K., Piston D.W, & Urano F. (2020). Wolfram syndrome 1 gene regulates pathways maintaining beta cell health and survival. Laboratory investigation; a journal of technical methods and pathology, 100(6), 849-862.
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