ProteoCool Pills#9: Use of low purity imidazole in buffer for IMAC may prevent protein detection during elution steps

Although imidazole itself should not give a UV280 signal, many commercially available products contain impurities which can give a strong UV signal.

Eg:   Absorbance spectrum of low purity imidazole buffered solution vs (black line) vs high purity imidazole (red line) acquired with nanodrop

The high imidazole concentration present in the IMAC Elution buffers (250-500mM) therefore the use of an imidazole with high Absorbance at 280nm may determine the detection of a plateau of absorbance during the elution (eg. Fig A) which may cover partially or completely the peak of eluted proteinFig.A:  His-Tagged protein purified from Expi293 surnantant with IMAC chromatography using an Elution buffer containing a low purity imidazole. 

                                                                    Solution (see Fig.B)

Be sure to use highly pure imidazole (eg Merck cod. 1.04716 ) which gives essentially no absorbance at 280 nm

Fig.B:  His-Tagged protein purified from Expi293 surnantant with IMAC chromatography using an Elution buffer containing an high purity imidazole. 

Acknowledgments:

" I would say a great thanks to Mirco Toccafondi, a great biochemist ever open to share his knowledge with the others !! "