Immuno-blotting for protein analysis has been described earlier [25 (link)]. Following protein separation through a gradient polyacrylamide gel (4–12%) and electro-blotting onto a PVDF membrane, proteins were identified and their expression rate determined by Western-blotting. The following proteins were detected: β-defensin-1, C1qR, ICAM1, ICOS, ICOSL, MYD88, and SOCS (all Abcam, Cambridge, UK). Details of dilution and producers are provided in Table 2 .
Membranes were blocked for 1 hour (room temperature) in PBS containing 0.01% Tween-20 and 2% bovine serum albumin. The primary antibodies were added at concentrations indicated inTable 1 and incubated overnight at 4°C. Following 3 washes with blocking buffer, membranes were incubated with secondary species specific antibodies labelled with horse radish peroxidase for 1 hour. Unbound antibody was washed off by 3 washes with blocking buffer and protein bands were visualised by exposure to X-ray films.
GAPDH expression was used to normalize protein expression and estimated changes induced by the different treatments.
Membranes were blocked for 1 hour (room temperature) in PBS containing 0.01% Tween-20 and 2% bovine serum albumin. The primary antibodies were added at concentrations indicated in
GAPDH expression was used to normalize protein expression and estimated changes induced by the different treatments.
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