Introduction

Samples were run on a gel and immunoblotted to demonstrate limits of 14-3-3 detection by anti 14-3-3.

Protocol

Protein preparation

Samples were prepared as serial dilutions in PBS (phosphate buffered saline. An equal volume of 2X denaturation gel loading buffer was added and samples were heated to 95°C for 5 mins. A total of 20µl of sample was loaded onto a 16% Tris-glycine gel that was run for 60 mins at 100 volts, 120mA.

Western Blotting and Visualisation

Gels were transferred onto PVDF and blocked o/n in 5% (in PBS)non-fat milk . Blots were incubated for 2hrs at room temp with 1^o antibody 1µg/ml anti 14-3-3 in PBS-Tween (0.05%). Blots were then washed 4x5mins in PBS-Tween and incubated for 1hr in 2^o antibody (peroxidase conjugated swine anti-rabbit 1:10,000) in PBS-Tween (0.05%). After 4 washes in PBS the blot was developed using ECL-plus and visualised by UVP image analysis.

Figure: Western Blot