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292 Tchaga
5. Use the BCA Protein Assay (Pierce; Cat. no. 23235) for protein quantitation.
6. Start preparing the columns while centrifuging the samples.
7. If extract or lysate is not translucent, you can clarify the sample by passing it
through a 0.45-μm filter or filter paper.
8. Use the BCA Protein Assay (Pierce; Cat. no. 23235) for protein quantitation.
9. We recommend a maximum sample load of 8 mg of total protein over a single
column. If loading higher amounts, additional washing steps should be performed.
Up to 5 ml of extract can be added to the column at a time. If your sample
volume is larger than 5 ml, then add the extract in steps.
10. This type of purification is referred to as mixed batch/gravity flow chromatography
in which the adsorption of the target proteins is carried under batch mixing
of the sample with the resin, followed by gravity-based adsorption, washing, and
elution.
11. Optional: If a cold room environment is not available perform this and the
following steps at room temperature, otherwise continue at 4ºC.
Acknowledgments
I thank Dr. Andrew Farmer for helping with the linguistic review of this
article.
References
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