Protein expression was evaluated by immunoblot. Samples were run in a PAGErTM Mini-gel Chamber (Lonza, Milan, Italy) using 10% acrylamide gels with a Tris-Glycine buffer and subsequently semi-dry blotted for 2 h with 50 mA current on PVDF membrane. After blocking for 1 h with 5% not-fat milk in Tween/Tris buffered salt solution (T-TBS), membranes were incubated overnight at 4 °C with primary antibodies (anti-actin 1:10000, anti-TTP 1:1000 Millipore, Milan, Italy, anti-14-3-3 1:1000 Abcam, Cambridge, United Kingdom and anti-TNF-α 1:6000, Inflectra, Pfizer, New York, United States). Membranes were then washed in T-TBS and incubated for 1 h at 37 °C with anti-rabbit HRP-conjugated secondary antibody 1:50000 (Millipore, Milan, Italy) and anti-human HRP-conjugated secondary antibody 1:10000 (Sigma-Aldrich, St. Louis, MO, United States). Chemiluminescence was developed using LiteAblot® TURBO (EuroClone®, Milan, Italy) and exposed on Kodak Biomax film (Sigma-Aldrich, St. Louis, MO, United States). Protein expression was quantified on immunoblot images using the ImageJ software and are reported in percentage respect to actin.
Pagertm mini gel chamber
Manufactured by Lonza
Sourced in Italy
The PAGErTM Mini-gel Chamber is a compact, gel electrophoresis system designed for the separation and analysis of macromolecules, such as proteins and nucleic acids. It provides a reliable and efficient platform for various research applications.
Automatically generated - may contain errors
2 protocols using pagertm mini gel chamber
1
Quantitative Immunoblot Analysis of Protein Expression
2
Immunoblotting for Protein Analysis
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Cells (1x10 7 ) were cultured as reported above, collected, washed in cold PBS and lysed using a lysis buffer composed by Tris-HCl 10 mM pH 7.4, EDTA 100 mM, NaCl 100 mM, SDS 0.1%, Protease inhibitor cocktail 1%. Samples were then run on 10% acrylamide gels in a Trys-Glycine buffer in a PAGEr TM Mini-gel Chamber (Lonza, Milan, Italy) and then semi-dry blotted for 2 h with 50 mA current on PVDF membrane. Membranes were blocked for 1 h with 5% not-fat milk in Tween/tris-buffered salt solution (TTBS) and incubated overnight at 4°C with primary antibodies (rabbit anti-actin 1:20000 and mouse anti-DDK 1:1000).
Membranes were then washed twice with TTBS and incubated for 1 h at 37°C with secondary anti-rabbit and anti-mouse HRP-conjugated antibodies, at 1:50000 and 1:25000 dilutions, respectively. Chemiluminescence was developed using LiteAblot ® TURBO kit following manufacturer's instructions and exposed on Kodak Biomax light film.
Membranes were then washed twice with TTBS and incubated for 1 h at 37°C with secondary anti-rabbit and anti-mouse HRP-conjugated antibodies, at 1:50000 and 1:25000 dilutions, respectively. Chemiluminescence was developed using LiteAblot ® TURBO kit following manufacturer's instructions and exposed on Kodak Biomax light film.
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