Editing 293E Protein Production (section)

==== <br>TROUBLESHOOTING  ====

Problem: Purification from IMAC does not work. 

[Step 13] 

Solution: Consider the following: 

1. Perform SDS-PAGE analysis of the feed, flowthrough, washes, and pooled fractions to see whether the r-protein was captured by the resin or not. If the His-tagged protein is in the flowthrough, the tag on the protein itself may be sterically hindered and inaccessible for capture by the column. 

2. The r-protein could be aggregated, thus hampering its capture onto the column. Check this by gel filtration chromatography. 

3. Increasing the pH of the feed to 8.0 or higher can enhance r-protein binding to the column, and charging the IMAC column with nickel instead of cobalt can increase binding affinity. 

4. Sometimes, the r-protein elutes in IMAC wash buffer 2. In this case, decrease the imidazole concentration of the wash buffer. 

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[[Category:293_Expression]] [[Category:Protocols]]