Western blot analysis was carried out as follows: protein extracts were boiled in loading buffer, separated by 10% SDS-PAGE, electrophoretically transferred onto a polyvinylidene fluoride membrane (Amersham Biosciences), blocked overnight at 4°C in blocking buffer (54 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Nonidet P-40, 0.05% Tween 20, 5% nonfat dry milk) and analyzed using primary antibodies followed by HRP-conjugated secondary antibody. Prestained Protein Ladder SM0671 (Fermentas) was used as a size marker. LumiGLO substrate (Kirkegaard & Perry Laboratories, Inc.) was used to develop the blot. Luminescence was captured using Amersham hyperfilm ECL films (Amersham-GE Healthcare Bio-Sciences, PA, USA).
Lumiglo substrate
Manufactured by LGC
LumiGLOTM substrate is a chemiluminescent substrate used for the detection of proteins in western blot analysis. It produces a luminescent signal when exposed to horseradish peroxidase (HRP), which is often conjugated to secondary antibodies in immunodetection procedures.
Automatically generated - may contain errors
4 protocols using lumiglo substrate
1
Western Blot Analysis Protocol
2
Cytokine Profiling with Protein Arrays
3
Cytokine Profiling with Protein Arrays
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The presence of selected cytokines was evaluated in samples of CM with human cytokine protein arrays (R&D Systems Inc.) according to the manufacturer’s instructions. Antigen-antibody complexes were visualized using LumiGLO substrate (Kirkegaard & Perry Laboratories, Inc.) and chemiluminescent sensitive film (Kodak). Densitometry was performed by image analysis (ImageJ) to estimate the amount of protein present in each sample.
4
Neutrophil Cytokine Secretion Analysis
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Secreted cytokines from neutrophils and IL‐8‐treated neutrophils were evaluated using mouse cytokine antibody array (ab133995, Abcam, Cambridge, MA, USA) according to the manufacturer's instructions. Antigen‐antibody complexes were visualized using LumiGLO substrate (Kirkegaard & Perry Laboratories, Inc.) and chemiluminescent sensitive film (Kodak). Densitometry was performed via image analysis (Image J) for quantification of cytokine secretion.
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