RASF were trypsinized and nuclear and cytoplasmic extracts prepared with NC-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) according to the manufacturers protocol. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane (Amersham HybondTM P; GE Healthcare). Membranes were blocked with 5% milk in TBS-T and incubated overnight at 4°C with rabbit anti-human PIWIL4 (PA5-31448; Thermo Scientific), rabbit anti-human PIWIL2 (sc-67303; Santa Cruz), rabbit anti-human Lamin B1 (ab16048; Abcam) and mouse anti-human α-tubulin (ab7291; Abcam) antibodies in 5% milk. HRP-labeled species-specific secondary antibodies (Jackson Immuno Research) were used and signals were detected using enhanced chemiluminescence Western BrightTM ECL (Advansta) and the Fusion FX imager (Vilber Lourmat). The Alpha Imager software system (Alpha Innotech) was used to analyse the results.
Hrp labeled species specific secondary antibodies
Manufactured by Jackson ImmunoResearch
HRP-labeled species-specific secondary antibodies are designed to detect the presence of primary antibodies in various immunoassays. These antibodies are conjugated with the enzyme Horseradish Peroxidase (HRP), which can catalyze a colorimetric reaction upon the addition of a suitable substrate, allowing for the visualization and quantification of the target analyte.
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Lab products found in correlation
2 protocols using hrp labeled species specific secondary antibodies
1
Extraction and Analysis of PIWIL Proteins
2
Western Blot Analysis of Cell Lysates
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Whole cell lysates were prepared by lysing the cells in Laemmli buffer. Proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked with 5% milk in TBS-T and incubated over night at 4°C with mouse anti-proliferating cell nuclear antigen (PCNA) (PC10, Novus Biologicals #NB500-106, 1:1000), mouse anti-tubulin (DM1A, Abcam, # ab7291, 1:10'000), rabbit anti-SIRT1 (E104, Abcam, #ab32441, 1:500) or mouse anti-Vimentin antibodies (Abcam, #ab7752, 1:1000) in 5% milk.HRP-labeled species specific secondary antibodies (Jackson ImmunoResearch, 1:10000) and enhanced chemiluminescence (GE Healthcare) were used for visualization.
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