Samples were run on a gel and immunoblotted to demonstrate
limits of 14-3-3 detection by anti 14-3-3.
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
antibody 1µg/ml anti 14-3-3 in PBS-Tween (0.05%).
Blots were then washed 4x5mins in PBS-Tween and incubated for 1hr
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
|Band on blot (left to right)
|Unpurified bacterial protein
|Not visible (no 14-3-3 cross reactivity)
|Unpurified 14-3-3 bacterial clone
|first visible band, note lower mwt band
|7.8125ng (weak band)
|3.90625ng (barely visible above