Total proteins (20 µg) were denatured at 70°C for 10 min, mixed with NativePAGE Sample Buffer (Thermo Fisher Scientific, Inc.), electrophoresed on Novex 4–12% Tris Glycine Midi Protein Gels (Thermo Fisher Scientific, Inc.) and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% milk for 1 h and incubated with anti-human CRP antibodies overnight: Mouse anti-CRP (1:500; clone, C6; catalog no. M86284M; Meridian Life Science, Inc., Cincinnati, OH, USA), Mouse anti-CRP (1:500; clone, C5; catalog no. M86005M; Meridian Life Science, Inc.) and Mouse anti-CRP (1:500; polyclonal; catalog no. ab52687; Abcam, Cambridge, MA, USA). The membranes were washed 3 times with TBST (1% serum albumin in 50 mM Tris-HCl, pH 7.4, containing 0.05% Tween-20) at room temperature and incubated with a horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:2,000; catalog no. sc-2005; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 1 h. Membranes were washed three times, visualized with SuperSignal West Pico Chemilluminescent substrate (Thermo Fisher Scientific, Inc.) for 5 min and exposed to X-ray film. Densitometry was performed using Image J 2.1.4.7 software (National Institutes of Health).
Supersignal west pico chemilluminescent substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States
SuperSignal West Pico Chemilluminescent substrate is a laboratory reagent used for the detection and quantification of proteins in Western blot analysis. It is a luminol-based substrate that produces a chemiluminescent signal upon reaction with horseradish peroxidase (HRP)-labeled antibodies. The intensity of the emitted light is proportional to the amount of target protein present in the sample.
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2 protocols using supersignal west pico chemilluminescent substrate
1
Western Blot Analysis of C-Reactive Protein
2
Western Blotting Protein Analysis
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Cell pellets were resuspended in PBS/glycerol (10% v/v) supplemented with EDTA free protease inhibitor cocktail set III (Calbiochem) and lysed by probe sonication for 3 x 10-20 second bursts.
Equal quantities of total protein were resolved on 8-12% w/v polyacrylamide gels and protein was transferred to Hybond TM -P PVDF membrane (Amersham Biosciences). Membranes were blocked with 5% w/v non-fat Marvel milk in TBST buffer (25 mM Tris pH 7.6, 0.15 M NaCl, 0.1% v/v Tween-20) at room temperature for an hour followed by incubation with primary antibodies (dilutions given in figure legends) at 4°C overnight. The membranes were then washed with TBST and incubated for 1 hour at room temperature with horseradish peroxidase conjugated secondary antibodies.
Following washing, the specific proteins were detected using a SuperSignal® West Pico Chemilluminescent Substrate (Thermo Scientific). Where band intensity was quantified, the density of unsaturated exposures was determined with Scion Image software (NIH, USA) after scanning the blots at 600 dpi resolution.
Equal quantities of total protein were resolved on 8-12% w/v polyacrylamide gels and protein was transferred to Hybond TM -P PVDF membrane (Amersham Biosciences). Membranes were blocked with 5% w/v non-fat Marvel milk in TBST buffer (25 mM Tris pH 7.6, 0.15 M NaCl, 0.1% v/v Tween-20) at room temperature for an hour followed by incubation with primary antibodies (dilutions given in figure legends) at 4°C overnight. The membranes were then washed with TBST and incubated for 1 hour at room temperature with horseradish peroxidase conjugated secondary antibodies.
Following washing, the specific proteins were detected using a SuperSignal® West Pico Chemilluminescent Substrate (Thermo Scientific). Where band intensity was quantified, the density of unsaturated exposures was determined with Scion Image software (NIH, USA) after scanning the blots at 600 dpi resolution.
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