For cytokine ELISA, lung lysates were prepared from middle sections of left lung from each mouse. Comparable portions of lung section were weighed and sonicated in HBSS (Sigma) supplemented with protease inhibition cocktail (Roche; 1 tablet for 10 ml of HBSS). Approximately 0.5 mg of tissue was sonicated as described higher up in methods in 500 μl of supplemented HBSS. 75 μl from each lysate was used for TNF and IL-β murine cytokine ELISAs (R&D).
Protease inhibition cocktail
Manufactured by Roche
Sourced in Germany
The Protease inhibition cocktail is a laboratory product designed to inhibit the activity of proteases, which are enzymes that can break down proteins. This product can be used to preserve the integrity of protein samples during various experimental procedures. The core function of the Protease inhibition cocktail is to provide a way to prevent unwanted protein degradation in a research or diagnostic setting.
Automatically generated - may contain errors
Lab products found in correlation
Hanks balanced salt solution (hbss), by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Roche (1 mentions)
Np40 lysis buffer, by Thermo Fisher Scientific (1 mentions)
Tissuelyser lt, by Qiagen (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Akt antibody, by Cell Signaling Technology (1 mentions)
Hrp conjugated goat anti rabbit igg secondary antibody, by OriGene (1 mentions)
Ecl detection kit, by Bio-Rad (1 mentions)
M iggκ bp hrp sc 516102, by Santa Cruz Biotechnology (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Gapdh antibody sc 47724, by Santa Cruz Biotechnology (1 mentions)
3 protocols using protease inhibition cocktail
1
Lung Cytokine ELISA Quantification
2
Western Blot Protein Analysis Protocol
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Cells and tissue were lysed with NP40 lysis buffer (Life Technologies) containing protease inhibition cocktail (Roche, Mannheim, Germany). Tissue was shredded by Tissue Lyser LT (Qiagen). Protein samples were loaded on a 4-12% SDS/PAGE gel and transferred onto a nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK). After blocking with 5% dry milk in TBST solution (TBS pH 7.2, 0.1% Tween-20), the membrane was incubated with primary antibody solution (diluted in 2 % dry milk in TBST, dilution factors are indicated in the following table) at 4°C overnight. Next day, after three time washing with 2% dry milk in TBST, the membrane was incubated with secondary antibody (horseradish peroxidase-conjugated antibodies (Cell Signaling, Danvers, MA, USA)) diluted in 2% dry milk in TBST for 1 hour at room temperature and finally washed three times. Signals were detected using a chemiluminescent kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
3
Protein Expression Analysis in Cardiac Tissue
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Left ventricular myocardial biopsies or cells were lysed in RIPA protein lysis buffer containing PMSF and protease inhibition cocktail (Roche Diagnostics GmbH, Mannheim, Germany) using the Bead Ruptor 4 Mini Homogenizer (Omni International). Protein concentration was determined with a BCA protein assay kit (Thermo Scientific, Rockford, USA). Protein extracts were separated on a polyacrylamide gel and subsequently transferred to a PVDF membrane. The membrane was blocked in 5% non‐fat dry milk/TBS‐T buffer followed by overnight incubation at 4°C with primary antibody. After rinsing, the membrane was incubated with HRP conjugated secondary antibody and detected by use of the ECL detection kit (Bio‐Rad). Densitometric quantification of protein bands was performed with the Chemidoc Touch Imaging System (Bio‐Rad). Following antibodies were used: anti‐SERPINA3 rabbit monoclonal antibody (ab205197, Abcam, Cambridge), Akt antibody (#9272) (Cell Signaling Technologies, Massachusetts), and HRP conjugated goat anti‐rabbit IgG secondary antibody (TA1300233, Origene). GAPDH was used to normalize for different protein content using GAPDH antibody (sc‐47724) and mIgG κ BP‐HRP (sc‐516102) (Santa Cruz Biotechnology, Inc, Dallas, Texas).
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