Difficult Protein Purification Columns

The Broad Institute
Gerstner Center for Cancer Diagnostics
Postdoctoral Researcher

Overall

Quality of Results

Ease-of-Optimization

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Questions/Comments

Company:

Thermo Scientific

Product Name:

Zeba Spin Desalting Columns (2mL)

Catalog Number:

89890

I am biotinylating and adding a terminal alkyne to a purified protein of interest (17 kDa) using NHS-ester chemistry. I needed to remove the remaining unreacted biotin and alkyne from the in-tact protein via these purification columns.

Experimental Design and Results Summary

Application

Protein purification

Starting Material

purified protein, Propargyl-PEG8-NHS, biotin-NHS

Tips

The columns must be taped into the tubes!

Results Summary

The columns kept on falling out of the tubes and snapping in the centrifuge, even when the centrifuge was put on a slow ramp up. The only way to fix this was to tape them into the conical collection tubes (photo attached). Even with that, I lost 50% of my protein yield after purification

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Summary

The Good

The columns did remove unreacted biotin and alkyne.

The Bad

I lost 50% of my protein and the tubes were difficult to work with.

The Bottom Line

These columns are difficult to use and lose protein, but they will get the job done.

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