Twenty μg of protein per well was subjected to electrophoresis on 4–12% NuPAGE Bis-Tris Gel in XCell SureLock Mini-Cell Units (Life Technologies, Budapest, Hungary). Proteins were transferred from gels to nitrocellulose membranes, using the iBlot Gel Transfer System (Life Technologies, Hungary). The antibody binding was detected with the WesternBreeze Chromogenic Western blot immundetection kit (Life Technologies, Budapest, Hungary). The blots were incubated on a shaker with α2A-AR, α2B-AR, α2C-AR and β-actin polyclonal antibody (Santa Cruz Biotechnology, California, 1:200) in the blocking buffer. Images were captured with the EDAS290 imaging system (Csertex Ltd., Hungary), and the optical density of each immunoreactive band was determined with Kodak 1D Images analysis software. Optical densities were calculated as arbitrary units after local area background subtraction.
Xcell surelock mini cell units
Manufactured by Thermo Fisher Scientific
Sourced in Hungary
The XCell SureLock Mini-Cell Units are a series of electrophoresis systems designed for the separation and analysis of biomolecules, such as proteins and nucleic acids. These units provide a controlled and consistent environment for performing electrophoresis experiments.
Automatically generated - may contain errors
3 protocols using xcell surelock mini cell units
1
Quantitative Western Blot Analysis
2
Western Blot Analysis of α2-Adrenergic Receptor Subtypes
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20 μg of protein per well was subjected to electrophoresis on 4%-12% NuPAGE Bis-Tris Gel in XCell SureLock Mini-Cell Units (Life Technologies) (n = 5 for each α2-AR subtype antagonists). Proteins were transferred from gels to nitrocellulose membranes, using the iBlot Gel Transfer System (Life Technologies). The antibody binding was detected with the WesternBreeze Chromogenic Western blot immundetection kit (Life Technologies). The blots were incubated on a shaker with α2A-AR, α2B-AR, α2C-AR, and β-actin polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:200) in the blocking buffer. Images were captured using the EDAS290 imaging system (Csertex Ltd, Budapest, Hungary), and the optical density of each immunoreactive band was determined with Kodak 1D Images analysis software. Optical densities were calculated as arbitrary units after local area background subtraction.
3
Quantifying Protein Expression in Cervical Tissues
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Cervical samples were stored at -70°C until Western blot studies. Cervices were powdered with a Sartorius Mikro Dismembrator U (Sartorius, Göttingen, Germany) and homogenized in a RIPA Lysis Buffer combined with PMSF solution, sodium orthovanadate solution and protease inhibitor cocktail solution (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). 50 µg of protein per well was subjected to electrophoresis on 4-12% NuPAGE Bis-Tris Gel in XCell SureLock Mini-Cell Units (Life Technologies, Budapest, Hungary). Proteins were transferred from gels to nitrocellulose membranes using the iBlot Gel Transfer System (Life Technologies, Budapest, Hungary). Antibody binding was detected with the WesternBreeze Chromogenic Western Blot Immundetection Kit (Life Technologies, Budapest, Hungary).
The blots were incubated on a shaker with RhoA, Roc1, Roc2 and β-actin monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:200) in the blocking buffer.
The optical density of each immunoreactive band was determined with Kodak 1D Images analysis software (Carestream Health, Inc., Rochester, NY, USA). Optical densities were calculated as arbitrary units after local area background subtraction.
The blots were incubated on a shaker with RhoA, Roc1, Roc2 and β-actin monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:200) in the blocking buffer.
The optical density of each immunoreactive band was determined with Kodak 1D Images analysis software (Carestream Health, Inc., Rochester, NY, USA). Optical densities were calculated as arbitrary units after local area background subtraction.
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