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Western blotting

UK Utsa Karmakar
JC Julia Y. Chu
KS Kruthika Sundaram
AA Anne L. Astier
HG Hannah Garside
CH Carsten G. Hansen
ID Ian Dransfield
SV Sonja Vermeren
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Neutrophils were lysed with ice-cold lysis buffer [20 mM Tris-HCl (pH 7.5), 150 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 0.1 mM PMSF, 10 μg/ml each of antipain, aprotinin, pepstatin A and leupeptin]. The detergent-soluble material was boiled in reducing sample buffer, subjected to SDS-PAGE and wet transferred to PVDF membrane (Millipore) for detection of proteins of interest using suitable antibodies. For IgG degradation assays, membranes were incubated with a HRP-coupled anti-rabbit secondary antibody. Blots were developed using chemoluminescence (Millipore).

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