Editing Protein purification using intein (section)

=== Elution of the Target Protein  ===

Following on-column cleavage the target protein is eluted from the column using the Column Buffer. The intein-CBD tag remains bound to the resin. Fraction sizes of about one third of the column bed volume typically result in the elution of the target protein within the first few fractions (sample 8). 

The protein concentration in each fraction can be determined by the Bradford Assay and the eluted fractions should be analyzed by SDS-PAGE. To check cleavage efficiency, remove 100 μl of resin and mix with 50 μl 3X SDS Sample Buffer. Boil for 5 minutes and spin the resin down. Analyze the supernatant (3-10 μl, sample 9) by SDS-PAGE. If a large amount of the precursor still remains uncleaved, continue incubation of the column for an additional 12-24 hours before conducting a second elution. 

When pTYB21 is used, a small peptide (1.6 kDa) is also cleaved from the intein tag and co-eluted with the target protein. Due to its small molecular weight, the cleaved peptide cannot be detected on a regular SDS-PAGE gel and can be removed by dialysis. 

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