Complete proteinase inhibitor mixture

Manufactured by Roche

Sourced in United States

Complete proteinase inhibitor mixture is a laboratory product that provides a broad range of protection against serine, cysteine, and metalloproteases. It is designed for use in protein extraction and purification procedures to prevent proteolytic degradation of target proteins.

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Proteinase inhibitors (217 citations) Complete proteinase inhibitor (54 citations) Proteinase inhibitor mixture (13 citations)

8 protocols using complete proteinase inhibitor mixture

Protein Extraction from Rat Hepatocytes

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The rat hepatocytes and liver tissue samples were lysed with ice-cold lysis buffer containing the following: 50 mmol/l Tris-HCl, pH 7.4; 1% NP-40; 150 mmol/l NaCl; 1 mmol/l EDTA; 1 mmol/l phenylmethylsulfonyl fluoride; and complete proteinase inhibitor mixture (one tablet per 10 ml; Roche Molecular Biochemicals, Indianapolis, IN, USA). The lysates were sonicated using the Sonicator VCX130 (Sonics & Materials) on ice, followed by centrifuging at 12000 g for 10 minutes at 4°C and the supernatants were retained. The protein concentration in the cell lysate was quantified using the DC protein assay kit (Bio-Rad). After determination of the protein content with the DC protein assay kit. Western blot analysis was performed.

Huang X., Gao Y., Qin J, & Lu S. (2014). The Role of miR-34a in the Hepatoprotective Effect of Hydrogen Sulfide on Ischemia/Reperfusion Injury in Young and Old Rats. PLoS ONE, 9(11), e113305.

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Protein Expression Analysis by Western Blot

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Cells were cultured and treated as described above and then lysed with ice-cold lysis buffer containing 50 mmol/L Tris-HCl, pH 7.4; 1% NP-40; 150 mmol/L NaCl; 1 mmol/L EDTA; 1 mmol/L phenylmethylsulphonyl fluoride; and complete proteinase inhibitor mixture (one tablet per 10 mL; Roche Molecular Biochemicals, Indianapolis, IN, USA). After protein content determination using a DC Protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA) and subsequently incubation with dilute solution (1 : 1000) of primary antibodies including anti-Bax, anti-Bcl-2, anti-caspase3, and anti-actin (Santa Cruz Biotechnology Inc, Santa Cruz, CA). The membranes were then exposed to the secondary antibodies, that is, alkaline phosphatase-labeled goat anti-rabbit immunoglobulin (Santa Cruz), at a dilution of 1 : 1000, followed by exposure to X-ray film [19 (link)]. The protein bands were detected using an enhanced chemiluminescence western blotting detection kit (Amersham, Little Chalfont, Buckinghamshire, UK).

Xie W., Xie Q., Jin M., Huang X., Zhang X., Shao Z, & Wen G. (2014). The β-SiC Nanowires (~100 nm) Induce Apoptosis via Oxidative Stress in Mouse Osteoblastic Cell Line MC3T3-E1. BioMed Research International, 2014, 312901.

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Western Blotting of Lysed Cells

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INS-1 cells and isolated rat islets were lysed with an ice-cold lysis buffer containing 50 mmol/l Tris-HCl, pH 7.4; 1% NP-40; 150 mmol/l NaCl; 1 mmol/l EDTA; 1 mmol/l phenylmethylsulphonyl fluoride; and a complete proteinase inhibitor mixture (one tablet per 10 ml; Roche Molecular Biochemicals, Indianapolis, IN, USA). After protein content determination using a DC protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA), western blotting was performed as described previously [37 (link)].

Yin Y., Yong W., Yu J., Zhang X., Lin H., Zhu Y, & Han X. (2016). Pdcd2l Promotes Palmitate-Induced Pancreatic Beta-Cell Apoptosis as a FoxO1 Target Gene. PLoS ONE, 11(11), e0166692.

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Jurkat T Cell Lysis Protocol

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Jurkat T cells were lysed with ice-cold lysis buffer containing: 50 mmol/l Tris–HCl, pH 7.4; 1% NP-40; 150 mmol/l NaCl; 1 mmol/l EDTA; 1 mmol/l phenylmethylsulfonyl fluoride; and complete proteinase inhibitor mixture (one tablet per 10 ml; Roche Molecular Biochemicals, Indianapolis, IN, USA). Protein concentration in the cell lysate was quantified using the DC protein assay kit. Following protein content determination, western blot analysis was performed.

Li A., Shuai X., Jia Z., Li H., Liang X., Su D, & Guo W. (2015). Ganoderma lucidum polysaccharide extract inhibits hepatocellular carcinoma growth by downregulating regulatory T cells accumulation and function by inducing microRNA-125b. Journal of Translational Medicine, 13, 100.

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Protein Extraction and Western Blot

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HepG2 cells, primary mouse hepatocytes and ob/ob liver tissue were lysed with ice-cold lysis buffer containing: 50 mmol/l Tris–HCl, pH 7.4; 1% NP-40; 150 mmol/l NaCl; 1 mmol/l EDTA; 1 mmol/l phenylmethylsulfonyl fluoride; and complete proteinase inhibitor mixture (one tablet per 10 ml; Roche Molecular Biochemicals, Indianapolis, IN, USA). Individual immunoblots were probed with a rabbit anti-SREBP-1c antibody diluted 1:1000; and mouse anti-α-tubulin antibody diluted 1:5000.

Yan C., Chen J, & Chen N. (2016). Long noncoding RNA MALAT1 promotes hepatic steatosis and insulin resistance by increasing nuclear SREBP-1c protein stability. Scientific Reports, 6, 22640.

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Metabolic Labeling and Immunoprecipitation of Sp1

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T24T cells were cultured in each well of six-well plate till 70%–80% confluence and the cell culture medium was replaced with 0.1% FBS DMEM and incubated for another 24 hours. The cells were then treated with 10μM of ISO diluted in 2% FBS methionine/cysteine free DMEM containing ³⁵S-labeled methionine/cysteine (250μ Ci per dish, Trans ³⁵S-label, ICN) for the indicated time periods. The cells were extracted with lysis buffer (Cell Signaling, Boston, MA, USA) containing complete proteinase inhibitor mixture (Roche, Nutley, NJ, USA). Total lysate of 500mg was incubated with anti-Sp1 antibody-conjugated agarose beads (R&D Systems, Minneapolis, MN, USA) at 4°C overnight. The immunoprecipitate was washed with the cell lysis buffer five times, and heated at 100°C for 5 min after final washing. The protein samples were then subjected to sodium dodecyl sulfate-polyacryl-amide gel electrophoresis analysis. ³⁵S-labeled Sp1 protein was imaging captured with the PhosphorImager (Molecular Dynamics, Kent, MI, USA).

Zeng X., Xu Z., Gu J., Huang H., Gao G., Zhang X., Li J., Jin H., Jiang G., Sun H, & Huang C. (2016). Induction of miR-137 by isorhapontigenin (ISO) direct targeted Sp1 protein translation and mediated its anti-cancer activity both in vitro and in vivo. Molecular cancer therapeutics, 15(3), 512-522.

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Protein Expression Analysis in Mouse Hepatocytes

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AML-12 cells, primary mouse hepatocytes and liver tissue from mice were lysed with ice-cold lysis buffer containing: 50 mmol/l Tris–HCl, pH 7.4; 1% NP-40; 150 mmol/L NaCl; 1 mmol/L EDTA; 1 mmol/L phenylmethylsulfonyl fluoride; and complete proteinase inhibitor mixture (one tablet per 10 mL; Roche Molecular Biochemicals, Indianapolis, IN, USA). Individual immunoblots were probed with a rabbit anti-Foxo1, rabbit anti-PEPCK and rabbit anti-G6Pase (all diluted 1:1000) and a mouse anti-β-Actin mAb (diluted 1:5000).

Yan C., Li J., Feng S., Li Y, & Tan L. (2018). Long noncoding RNA Gomafu upregulates Foxo1 expression to promote hepatic insulin resistance by sponging miR-139-5p. Cell Death & Disease, 9(3), 289.

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Western Blotting Assay for Protein Expression

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INS‐1 cells were harvested with ice‐cold lysis buffer supplemented with complete proteinase inhibitor mixture (Roche). Other specific information of Western blotting assay was performed as previously described previously.¹⁹ The antibodies used are as follows: rabbit anti‐CASK antibody (1:1000; Cell Signaling), rabbit anti‐DNMT1 (1:1000; Cell Signaling), mouse anti‐DNMT3a (1:1000; Santa Cruz Biotechnology), rabbit anti‐DNMT3b (1:1000; Abcam), rabbit anti‐Akt(pan) antibody (1:1000; Cell Signaling), rabbit anti‐p‐Akt Ser473 antibody (1:5000; Abcam), rabbit anti‐iNOS Antibody (1:1000; Cell Signaling) or rabbit anti‐β‐tubulin antibody (1:4000; Cell Signaling).

Wang T., Liu X., Xie J., Yuan Q, & Wang Y. (2020). Cask methylation involved in the injury of insulin secretion function caused by interleukin1‐β. Journal of Cellular and Molecular Medicine, 24(24), 14247-14256.

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