Table 5.

Troubleshooting Possible Problems

Protocol	Problem	Possible Cause		Solution
1,2	No high molecular weight crosslinking bands or smearing observed in the experimental lane compared to the negative control lanes.	Low abundance of substrate of interest		Use more lysate or substrate of interest; if using endogenous substrate, consider use of overexpressed-tagged substrate
		Good abundance of substrate of interest	Poor antibody sensitivity	Optimize the Western blot protocol by testing different primary or secondary antibody dilutions or using a different primary antibody
			Low lysate concentration in the crosslinking reaction	Use a more concentrated lysate in the crosslinking reaction to increase the likelihood for kinase-substrate interactions.
			Low ATP-arylazide concentration in crosslinking reaction	Increase the final concentration of ATP-arylazide in the kinase reactions
			ATP-arylazide degradation, especially if older than 6 months	Use a freshly dissolved stock of ATP-arylazide
			expired kinase buffer	Make fresh 10X kinase buffer
1,2	High background in the negative control lanes, which could mask crosslinked bands in the crosslinking samples	The substrate of interest is highly abundant in the lysate		Decrease the amount of lysate used for the kinase-catalyzed crosslinking experiments
		Too much HRP developer used		Dilute the HRP developer before adding to membrane
				Use less sensitive fluorescence detection
		Too long of an exposure time on the imager		Shorten the length of exposure
		The blocking step was unsuccessful		Optimize the blocking step by trying alternative blocking reagents and incubation times
2	Low levels or undetectable levels of the substrate of interest by Western blot after immunoprecipitation	Immunoprecipitation step was unsuccessful		Use a larger quantity of primary antibody
				Use a different immunoprecipitation-compatible primary antibody
				Express the substrate with an immunoprecipitation-compatible tag
				Use a different enrichment method
2	Unequal substrate protein levels visualized by Western blot after immunoprecipitation	Accidently removed beads when performing washing steps		During the washing steps, remove small amounts of buffer sequentially to avoid removing beads
2	Unable to distinguish substrate of interest from the antibody bands on the Western blot after immunoprecipitation	The substrate of interest and the heavy chain (50 kDa) or light chain (25 kDa) of the antibody have similar molecular weights		Use either a light chain-specific or heavy-chain specific secondary antibody to visualize the Western blot