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Supersignal west pico enhanced chemiluminescence kit

Manufactured by Thermo Fisher Scientific
Sourced in United States

The SuperSignal West Pico enhanced chemiluminescence kit is a reagent system designed for the detection of target proteins in Western blot analysis. It utilizes a proprietary luminol/enhancer solution to produce a chemiluminescent signal in the presence of the target protein, allowing for sensitive visualization of the protein of interest.

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3 protocols using supersignal west pico enhanced chemiluminescence kit

1

Western Blot Analysis of Nectin-1 in Mouse Brain Lysates

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Mouse brains were minced and lysed in Pierce RIPA buffer (Thermo Fisher Scientific, Rockford, IL, USA) supplemented with protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL, USA). Tissue lysate was collected by centrifugation and quantified using Pierce BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). An equal amount of lysate was boiled in loading buffer and subjected to SDS-PAGE. Subsequently, the separated protein in gel was transferred onto the PVDF membrane, which was then blocked in 5% milk and incubated with anti-nectin1 mouse monoclonal antibody (sc-21722, Santa Cruz Biotechnology, Dallas, TX, USA) at 4 °C overnight. The membrane was washed and further incubated with HRP-conjugated goat-mouse IgG secondary antibody. After thorough washing, the target protein was imaged using a SuperSignal West Pico enhanced chemiluminescence kit (Thermo Fisher Scientific, Carlsbad, CA, USA). The same blots were probed with a β-actin rabbit monoclonal antibody (13E5, Cell Signaling Technology, Danvers, MA, USA) as a loading control.
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2

Monocyte Protein Extraction and Western Blot Analysis

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Protein extracts were prepared from treated CD14+ monocytes using cell extraction buffer supplemented with a protease inhibitor cocktail, containing phenyl-methyl-sulfonyl fluoride and phosphatase inhibitors. Protein concentrations were measured with BCA protein assay kits. Each well of SDS gel was loaded with 20 μg of protein and were electrophoresed at a constant 100 V in 1× Tris glycine buffer for 60 minutes. Proteins in gels were transferred to the PVDF membrane using wet transfer. After appropriate antibody treatments, the bands were visualized with SuperSignal West Pico enhanced chemiluminescence kit from Thermo Scientific (Rockford, Illinois).
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3

Western Blot Analysis of HIV-1 Proteins

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The Western blot protocol was described previously [26, 28] . Briefly, the cells were washed once with ice-cold phosphate-buffered saline and harvested with lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA). Cellular debris was removed by centrifugation at 15000 g for 10 min. After centrifugation, the supernatant proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (EMD Millipore Corporation, Billerica, MA, USA). The Pierce Microplate BCA Protein Assay kit-Reducing Agent Compatible kit (Thermo Scientific, Inc., Rockford, IL, USA) was used to standardise the protein concentration in all samples. The membrane was probed with respective antibodies and immunoreactive proteins were visualised using the SuperSignal West Pico enhanced chemiluminescence kit (Thermo Scientific, Inc.). To detect HIV-1 proteins, the cell lysates were subjected to immunoblotting using sera collected from AIDS patients [20, 26] .
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