We used the previously described method to extract proteins from the worms and perform Western blot analysis [31 (link)]. Protein was extracted from frozen worm pellets using Fastprep24 (MP Biomedicals LLC, Solon, OH, USA) and PBS containing protease inhibitors. The extract was boiled with sample buffer containing sodium dodecyl sulfate (SDS) for 10 min and separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane. After reacting with the primary antibody overnight, the position and intensity of human α-synuclein were determined by use of horseradish peroxidase (HRP)-conjugated secondary antibody (PerkinElmer Inc., Boston, MA, USA) and the Amersham enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ, USA) and BioSpectrum imaging system (UVP, Upland, CA, United States). Human α-synuclein monoclonal antibody (sc-12767) and β-actin (sc-47778) antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Horseradish peroxidase hrp conjugated secondary antibody
Manufactured by PerkinElmer
Sourced in United States
Horseradish peroxidase (HRP)-conjugated secondary antibody is a type of laboratory reagent used in various immunoassay techniques. The HRP enzyme is covalently linked to the secondary antibody, which recognizes and binds to the primary antibody. This conjugate can then be used to detect the presence and quantity of a specific target analyte in a sample.
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Lab products found in correlation
β actin sc 47778 antibody, by Santa Cruz Biotechnology (1 mentions)
Amersham enhanced chemiluminescence system, by Cytiva (1 mentions)
Fastprep 24, by MP Biomedicals (1 mentions)
Pvdf membrane, by Cell Signaling Technology (1 mentions)
Anti ck2α, by Abcam (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
2 protocols using horseradish peroxidase hrp conjugated secondary antibody
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of SRF Phosphorylation
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The cells were lysed in a RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, and 1% IGEPAL CA-630, pH 7.4) with Protease Inhibitor Cocktail Set I (Calbiochem) and the protein Phosphatase Inhibitor Cocktail Set IV (Calbiochem). Equal amounts of lysate samples (20 μg of protein) were subjected to 10% SDS-PAGE and transferred to PVDF membranes (Millipore). The PVDF membranes were blocked with 2% BSA in 0.05% Tween 20 Tris-buffered saline buffer before incubation with one of the following primary antibodies: Anti-pS103SRF (1:1000; Cell Signaling), anti-SRF (1:1000; Millipore), anti-CK2α (1:4000; Abcam), or anti-β-actin (1:10,000; Millipore). A horseradish peroxidase (HRP)-conjugated secondary antibody (Perkin Elmer) was used for all Western blots. The protein bands were visualized using a chemiluminescent HRP substrate and the X-Lite Imaging System (Avegene, Taipei, Taiwan). The density of each band was quantified using ImageJ (NIH).
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