Table 2.

Troubleshooting

Step	Problem	Reason	Solution
24	Poor cell recovery	The cells were not pelleted sufficiently during the centrifugation.	Increase the time or speed of the centrifugation. Be sure wash solution contains BSA.
40	Cell lysate is cloudy after sonication	Sonication did not completely lyse cells or SDS-protein complexes precipitate from solution.	Increase sonication times and be sure to avoid foaming of samples. Ensure the proper volume of lysis buffer was used in Step 39.
56	White precipitate layer observed above beads after centrifugation of biotin captures	Lipids from cell membranes were not properly pelleted after sonication	Make certain that lysate is clear after sonication and centrifugation. If white layer is observed on top of cell lysate, remove lysate, and clear again by centrifugation.
64, Elution Option B, xviii	No pellet is observed after air drying the TCA concentrated iPOND eluate	Sample was lost during TCA precipitation	Centrifuge the supernatant saved in Step xiv. Proceed with steps xv-xviii. If no pellet is observed, centrifuge supernatant previously saved in Step xvii. Continue with step xix.
68	High background signal in the control sample	Protein binds to streptavidin beads non-specifically.	Use elution option B, increase the number of washes in Steps 62-63.
68	Poor signal for control proteins like PCNA in the experimental sample	Poor EdU incorporation.	Increase the number of cells used in each sample and ensure the cells are growing well prior to experiment.
68	Poor detection of protein of interest in the input samples	Poor antibody or formaldehyde crosslinking interferes with epitope detection.	Optimize immunoblotting conditions or change antibody. Consider increasing the boiling time in Step 65Aiii or Step 65Bxx to completely reverse the formaldehyde crosslinks.