The most obvious explanation is that the protein was not purified to homogeneity by the method employed.

The multiple bands may represent proteolytic products of pfu due to failure to use effective and sufficient amount of protease inhibitors in the set up to purification.

If you have purified Pfu (Polymerase from Pyrococcus furiosus) and are observing multiple bands in the Pfu lane after treatment in the heat block, several factors could contribute to the appearance of multiple bands. Here are some potential explanations:

To troubleshoot and identify the cause of the observed multiple bands, consider the following steps:

• Assess Pfu Purity: Reassess the purity of your purified Pfu sample using methods such as gel electrophoresis, spectrophotometry, or fluorometry to check for contaminants.
• Optimize PCR Conditions: Review and optimize your PCR conditions, including primer design, annealing temperature, and the number of cycles. Adjusting these parameters may help reduce nonspecific amplification.
• Evaluate Heat Treatment: Ensure that the heat treatment is sufficient to denature the Pfu enzyme. You may need to adjust the temperature and duration of the heat treatment.
• Verify Reaction Components: Check the quality and purity of all PCR reagents to eliminate the possibility of contaminants contributing to multiple bands.

By systematically investigating these factors, you should be able to identify and address the source of the multiple bands in the Pfu lane.