Table 1.

Troubleshooting table.

step	problem	possible reason	solution
4	Low yield of alcohol 2	Ethylenediamine is old Reaction interfered with by O2 and moisture Product destroyed during reaction quenching	Use either re-distilled ethylenediamine or fresh out of the bottle Make sure that the reaction is protected under dry N2 or Ar The reaction quenching is exothermic; add the quencher (1 N HCl) slowly and make sure that the flask is well cooled
7	Low yield of aldehyde 3	Aldehyde 3 is volatile	Avoid excess heating (above 35 °C)of the rotary evaporator water bath
11A(iv)	Low yield of HNE(alkyne)	HNE(alkyne) is not stable	It is prone to air oxidation and polymerization. Carry on nonstop through steps involving reaction workup, rotary evaporator, chromatography, to final storage
11B(viii)	Product 7 partially deprotected	One of the isomers of product 7 is not stable	Product 8 (deprotected 7) can be separated from 7 by chromatography on silica gel, or it can be carried through to the next step
30	Low yield of 14	Reaction yield can be reduced by adventitious O2 and/or moisture	Make sure that all the solvents are dry and that the reaction is protected under dry N2 or Ar. Thoroughly degas the solvents used
31	Incomplete deprotection of 14	Some of the catalyst (10% Pd/C) may be deactivated	Add more catalyst (10% Pd/C) and extend the reaction time. Typically, high catalyst loading can be tolerated. Ensure that the reaction is tightly sealed and that an atmosphere of hydrogen is maintained throughout
36	Difficult to remove impurities from HtPHA	Polarity of impurities is close to that of HtPHA	Run flash chromatography with gradient eluent, from 1:3 to 1:5 (vol/vol, hexanes: EtOAc)
	Final product decomposition	Light-induced photo- uncaging of the final product	During the synthesis (especially from 14 to 15, and 15 to HtPHA), protect the reaction flask from stray light
40A(x)	No Cy5 signal on gel	Failure of click coupling	Check lysate protein concentration. The concentration should be around 1.0 mg/ml Check each reagent in the click coupling step. Make sure that all of them are freshly prepared and that the concentrations are correct. Mix the reaction well Use cells treated globally with HNE(alkyne) as a positive control
	High Cy5 background	Old SDS-PAGE running buffer	Use fresh SDS-PAGE running buffer Rinse the gel several times and analyze the gel after each rinse to obtain the optimal result with highest signal-to-noise ratio Let the dye font run out completely before imaging the gel
	Incomplete TEV cleavage	Loss of TEV activity	Use a fresh aliquot. Avoid multiple freeze–thaw cycles Mix well after adding His6-TEV-S219V Increase TEV amount and incubation time for TEV cleavage
40B(xxviii)	Low yield of pulldown protein	Affinity protein purification condition is not optimal Protein is not eluting or eluting prematurely Protein is unstable after modification103	This protocol is optimized for His6-Halo-TEV-Keap1. Further optimization may be required for other proteins Monitor the protein in washes through SDS–PAGE. If premature elution is observed, decrease the concentration of imidazole in the wash buffers. Conversely, if no elution is observed, increase imidazole concentration to 200–400 mM or elute with Laemelli buffer to validate binding Add a proteasome inhibitor (bortezomib) in the lysis buffer and/or to cells
	Modification not found	Targeting is not efficient. Modification is reduced Inefficient MS conditions	Check modification of protein by Cy5 labeling, as described in Step 3A Make sure that TCEP is used instead of DTT in the sample preparation Adjust the ionization temperature to obtain optimal results. A low ionization temperature may lead to poor ionization and decrease sensitivity. A high ionization temperature may cause loss of modification
40C(xi); 40D(viii)	Low Firefly/Renilla/ GFP signal intensity	Significant loss of cells during rinsing Excessive cell death Low transfection efficiency Instrument setting is not optimal Response timing is not optimal	Perform the rinsing with care. Add the medium slowly along the side wall of the culture dish. Adding and removing medium should be done as gently as possible but also as efficiently as possible: wash plates sequentially (2 or 3 plates at a time) to avoid drying out. Mark the position on the plate where cells are washed Use the suggested cell density for transfection. If using a different transfection reagent or different cells, optimize transfection conditions Optimize transfection conditions for the cell type and the reagent used Run a positive control. For flow cytometry, cells transfected with GFP can be used. Adjust gain (i.e., laser power) if necessary Set up a time course for measurements (recommended time points for pilot trials: 4 h, 12 h, 18 h post T-REX light exposure)
	Batch-to-batch variability in targeting/ARE results	Difference in experimental setup and execution	Count cells and seed the numbers as specified in the protocol. Transfect and perform experiments at similar confluence Use cells at lower passage number (lower than 6–7 continuous passages)
	Activation of ARE-luciferase with light alone or with pre-HNE alone	Release of pre-HNE due to stray light Stressed cells	Protect photocaged-precursor-treated samples from stray light Count cells before seeding. A cell density that is too low or too high can stress the cells, leading to higher background