Briefly, approximately 35 µg of protein was run on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride membrane (PVDF, Biorad, USA). The membranes were blocked with skimmed milk (Merck, USA) and polyclonal antiPLCζ antibody (1:32000, Covalab, France), polyclonal anti-PAWP antibody (1:5000, abcam, UK) and monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), clone 6C5 (1:5000, Millipore, USA), were used as specific primary antibodies. After three times washing, the secondary antibodies, used for PAWP and PLCζ, were horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and for GAPDH was anti-rabbit IgG (all purchased from Dako, Japan). After three times washing, target proteins band were detected with an Amersham ECL Advance Western Blotting Detection Kit (GE Healthcare, Germany). The fire reader (Uvitec, UK) was used for recording chemiluminescence images. Densitometric analysis of the images was performed by Quantity One Software v 4.6.9 (Bio-Rad, Germany). Results were expressed as mean relative intensity (mean intensity of the patient’s band/mean intensity of fertile bands) (17 (link)). Figure 1 showed Western blot of PLCζ and PAWP in infertile men with globozoospermia (n=4) and fertile men (n=5).
Fire reader
Manufactured by Uvitec
Sourced in United Kingdom
The Fire Reader is a compact and versatile laboratory equipment designed for accurate and efficient analysis. It utilizes advanced technology to perform various measurements and data collection tasks within the laboratory setting. The core function of the Fire Reader is to provide reliable and consistent results for research and testing purposes.
Automatically generated - may contain errors
Lab products found in correlation
Skimmed milk, by Merck Group (1 mentions)
Amersham ecl advance western blotting detection kit, by GE Healthcare (1 mentions)
Gapdh clone 6c5, by Merck Group (1 mentions)
Quantity one software v4, by Bio-Rad (1 mentions)
Horseradish peroxidase hrp conjugated goat antimouse igg, by Agilent Technologies (1 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions)
2 protocols using fire reader
1
Western Blot Analysis of PLCζ and PAWP in Globozoospermia
2
Investigating Nucleic Acid Interactions
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Experiments were performed in 10 mM PBS buffer with 137 mM NaCl, pH 7.4. HDP (1 μM) was analyzed alone or mixed with HAP (1 μM) and target miRNA-373 (1 μM) for 1 h at 37 °C. The final volume of the solution was 50 μL. The sample was loaded onto a 4.0% agarose gel to separate the HDP-HAP complex from the substrate. 1 × tris-acetic-EDTA (TAE) buffer (90 mM tris, 90 mM acetic acid, and 10 mM EDTA, pH 8.0) was used as the running buffer. The gel was run for 1 hour at a constant current of 50 mA and the gel image was acquired with a gel-imaging system (UVItec FireReader, UK).44
Optimized HAP experiments were performed in 10 mM PBS buffer with 137 mM NaCl, pH 7.4. HAP-A, B, C, D and E (1 μM) was analyzed alone or mixed with a linker (1 μM) for 1 h at 37 °C. The final volume of the solution used to separate the HDP-HAP complex from the substrate was 50 μL and the other experimental conditions were the same as in the above protocol.
Amplified experiments were performed in 10 mM PBS buffer with 137 mM NaCl, pH 7.4. HAP-D (1 μM) was analyzed mixed with HDP (1 μM) and different concentrations of target miRNA-373 (1000, 100, 10, 1, 0.1, 0.01 and 0.001 nM) for 1 h at 37 °C. The final volume of the solution used to separate the HDP-HAP complex from the substrate was 50 μL and the other experimental conditions were the same as in the above protocol.
Optimized HAP experiments were performed in 10 mM PBS buffer with 137 mM NaCl, pH 7.4. HAP-A, B, C, D and E (1 μM) was analyzed alone or mixed with a linker (1 μM) for 1 h at 37 °C. The final volume of the solution used to separate the HDP-HAP complex from the substrate was 50 μL and the other experimental conditions were the same as in the above protocol.
Amplified experiments were performed in 10 mM PBS buffer with 137 mM NaCl, pH 7.4. HAP-D (1 μM) was analyzed mixed with HDP (1 μM) and different concentrations of target miRNA-373 (1000, 100, 10, 1, 0.1, 0.01 and 0.001 nM) for 1 h at 37 °C. The final volume of the solution used to separate the HDP-HAP complex from the substrate was 50 μL and the other experimental conditions were the same as in the above protocol.
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