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  • Coomassie Gel
 

Coomassie Gel

Introduction
Samples were run on a gel and coomassie brilliant blue stained to demonstrate protein purity.

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.

Coomassie gel and Visualisation
Gels were stained with coomassie brilliant blue, washed with destain and visualised by UVP image analysis

Figure: Coomassie gel


Sample Band on blot (left to right)
Protein MWT ladder .
Unpurified bacterial protein Note absence of 30kDa band
Unpurified 14-3-3 bacterial clone fraction note strong ~30kDa 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 (weak band)
HPLC purified 7.8125ng (v.v.weak)
HPLC purified 3.90625ng (barely visible above background)

coomassie of 14-3-3

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