Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in 4.5% stacking and 12% running gel. Cockroach or cricket brains were dissected out in PBS. After dissection, two brains were transferred into 50 μl of lysis buffer (57.7 mM Tris-base, 10% glycerine, and 2% SDS) containing 1.5 μl ß-mercaptoethanol (Z523A, Promega, WI, USA), and immediately homogenized. The homogenate (containing two brains) was heated for 3 min at 100°C, transferred on ice and then centrifuged. The supernatant was subjected to SDS-PAGE and blotted on a Hybond-P PVDF membrane (RPN303F, GE Healthcare UK Ltd, Little Chalfont, Bucks, UK). As a molecular weight marker, Dr. Western (Oriental Yeast Co., Ltd, Tokyo, Japan) was used. After blocking the membrane with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST, pH 7.6) for 30 min, it was incubated with the rabbit anti-tyrosine hydroxylase (1: 5,000) in TBST for 2 h at 25°C. The membrane was rinsed in four changes of TBST, and incubated in donkey anti-rabbit IgG conjugated to HRP (1:2,000; NA934V, GE Healthcare UK Ltd) in TBST for 90 min at 25°C. After being rinsed in four changes of TBST, the ECL Western Blotting Starter Kit (RPN2108, GE Healthcare UK Ltd) was used to visualize immunoreactivity.
Ecl western blotting starter kit
Manufactured by GE Healthcare
Sourced in Japan
The ECL Western Blotting Starter Kit is a laboratory equipment product designed for protein detection and analysis. It contains the necessary components for performing enhanced chemiluminescence (ECL) Western blotting techniques.
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3 protocols using ecl western blotting starter kit
1
Cockroach/Cricket Brain Protein Analysis
2
Quantifying Outer Membrane Vesicle Production
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Five microliter samples of the isolated OMVs or the E. coli cells of each strain were analyzed by SDS-PAGE with Coomassie Blue staining. OMV production was quantified as previously described 13 with some modifications. The SDS-PAGE band at ~ 37 kDa was analyzed by densitometry (Image J software, NIH, Bethesda, MD) to index the OMV concentration. The index was normalized against the OMV productivity of the wild type strain.
For western blotting, protein was transferred from the gel to a membrane sheet of Hybond P (GE Healthcare Ltd., Buckinghamshire, England) by the semi-dry transfer method. Hybridization was conducted using an anti-GFP antibody conjugated with horseradish peroxidase (Medical & Biological Laboratories Co., Nagoya, Japan) and an ECL Western Blotting Starter Kit (GE Healthcare Ltd.) following the manufacturer's protocol. The hybridization signals were detected by a ChemiDoc imaging system (Bio-Rad Laboratories Inc., Hercules, CA). The western blotting band was analyzed by densitometry to index the target protein expression. This index was normalized against the OMV productivity of the wild type strain.
For western blotting, protein was transferred from the gel to a membrane sheet of Hybond P (GE Healthcare Ltd., Buckinghamshire, England) by the semi-dry transfer method. Hybridization was conducted using an anti-GFP antibody conjugated with horseradish peroxidase (Medical & Biological Laboratories Co., Nagoya, Japan) and an ECL Western Blotting Starter Kit (GE Healthcare Ltd.) following the manufacturer's protocol. The hybridization signals were detected by a ChemiDoc imaging system (Bio-Rad Laboratories Inc., Hercules, CA). The western blotting band was analyzed by densitometry to index the target protein expression. This index was normalized against the OMV productivity of the wild type strain.
3
Quantification of GFP and Tsf-GFP in Floc Samples
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A floc sample (5 μL) was subjected to SDS-PAGE. For western blotting, proteins were transferred from the gel to a membrane sheet of Hybond P (GE Healthcare Ltd., UK) using the semi-dry transfer method. Hybridization was conducted using an anti-GFP primary antibody (Medical & Biological Laboratories Co., Japan) and an ECL Western Blotting Starter Kit (GE Healthcare Ltd.) according to the manufacturers' protocols.
Hybridization signals were detected using a ChemiDoc imaging system (Bio-Rad Laboratories Inc., Hercules, CA).
The protein bands were analyzed by densitometry (Image J software; NIH, Bethesda, MD) as an index of target protein expression. The amounts of GFP and Tsf-GFP were determined using purified GFP and Tsf-GFP as standards. Purification of GFP and Tsf-GFP was conducted using a Bio-scale Mini Profinity IMAC cartridge and Profinia protein purification system (Bio-Rad Laboratories Inc.) after ultrasonication of E. coli cells expressing each protein. Finally, the percentages of GFP and Tsf-GFP were determined based on total floc protein determined by the BCA method.
Hybridization signals were detected using a ChemiDoc imaging system (Bio-Rad Laboratories Inc., Hercules, CA).
The protein bands were analyzed by densitometry (Image J software; NIH, Bethesda, MD) as an index of target protein expression. The amounts of GFP and Tsf-GFP were determined using purified GFP and Tsf-GFP as standards. Purification of GFP and Tsf-GFP was conducted using a Bio-scale Mini Profinity IMAC cartridge and Profinia protein purification system (Bio-Rad Laboratories Inc.) after ultrasonication of E. coli cells expressing each protein. Finally, the percentages of GFP and Tsf-GFP were determined based on total floc protein determined by the BCA method.
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