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
| Sample |
Band on blot (left to right) |
| Unpurified bacterial protein |
Not visible (no 14-3-3 cross reactivity) |
| Unpurified 14-3-3 bacterial clone
fraction |
first visible band, note lower mwt band |
| HPLC purified |
1000ng |
| HPLC purified |
500ng |
| HPLC purified |
250ng |
| HPLC purified |
125ng |
| HPLC purified |
62.5ng |
| HPLC purified |
31.25ng |
| HPLC purified |
15.62ng |
| HPLC purified |
7.8125ng (weak band) |
| HPLC purified |
3.90625ng (barely visible above
background) |
