Immunoblotting was carried out according to Huang et al. (2007) (link) and Guccione et al. (2010) (link) with slight modifications: cell-free extracts or periplasmic samples were denatured by boiling in Laemmli sample buffer and separated by SDS-PAGE on 8 % (w/v) acrylamide gels and electroblotted onto nitrocellulose membrane (Hybond-C extra; GE Healthcare). Blocking was carried out with Gelatin-NET at room temperature for 1 h. A buffer containing 0.2 % (w/v) BSA and 2 % (w/v) polyvinylpyrolidone (PVP; Mw 24 000– 40 000) in PBS/Tween-20 (PBST) was used for membrane washing and in the dilution of anti-MfrA (Guccione et al., 2010 (link)), anti-GroEL and horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma). The HRP-conjugated bound antibodies were detected using the enhanced chemiluminesence (ECL) kit from GE Healthcare.
Enhanced chemiluminesence ecl kit
Manufactured by GE Healthcare
The Enhanced Chemiluminescence (ECL) kit is a laboratory reagent designed to facilitate the detection of proteins in western blotting procedures. The kit contains the necessary solutions to generate a luminescent signal when specific proteins are present, allowing for their visualization and quantification.
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2 protocols using enhanced chemiluminesence ecl kit
1
Immunoblotting with Modifications
2
Haemoglobin-Associated Peroxidase Detection
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Proteins were either denatured normally by boiling in Laemmli sample buffer containing both SDS and mercaptoethanol (for Coomassie staining) or were incubated for 1 h at 42°C in SDS sample buffer without mercaptoethanol (for haem detection). Proteins were separated by Tricine-SDS-PAGE and either stained with Coomassie blue G250 or electroblotted onto nitrocellulose membrane (Hybond-C extra, GE Healthcare). Covalently bound haem was detected as haem-associated peroxidase activity using the enhanced chemiluminesence (ECL) kit from GE Healthcare with X-ray film detection (Feissner et al., 2003) .
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