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Purification of GFPmut3b

Aims

To purify GFPmut3b protein to allow us to construct a calibration curve for the anaylsis of our data.

Materials and Methods

Link to the Protocol

Results

Figure 1.1. SDS-PAGE Gel (12%) of purification fractions. The picture is of an SDS-PAGE Gel of the fractions from the nickel affinity column. 2ul of fractions were laoded with 8ul water and 2ul of loading buffer. Sample M is a marker, Sample A is the fraction run off and Sample B and C are fractions 16 and 17 respectivly. It can be seen that fraction C contains a large band around 27kDa which corresponds to the GFPmut3b.

Discussion

Figure 1.1 shows that the GFPmut3b was purified and eluted in fraction 17. It can be seen that there is a very large band that corresponds to the GFPmut3b. In addition, when the fractions were collected fraction 17 had an intense green color. This confirmed that the GFPmut3b purified in fraction 17 was functional. A Bradford assay was carried out on fraction 17 giving an approximate concentration of 1mg/ml for ~2ml.

Conclusion

The GFPmut3b has been successfully purified in the functional form to a concentration of 1mg/ml. This will enable us to construct a calibration curve for our in vitro chassis to correlate fluorescence with the number of molecules of GFPmut3b.