We do a lot of bioconjugation chemistry (click chemistry in particular but also NHS and Maleimide chemistries). Our method to valid the conjugation reactions have been to use SDS-PAGE gels followed with densitometry. However one particular challenge with such analyses is that proteins conjugated with non-proteins like DNA or PEG migrate with an inconsistent MW and also in a smear making it hard to isolate the relevant bands. I assume that this has to do with having a poly-disperse radius of gyration. Are there tricks that I can do to get tighter bands?

  • $\begingroup$ where would the poly-disperse radius of gyration arise from if the molecules are coated with SDS? $\endgroup$ – Ben Jun 4 '12 at 11:27
  • $\begingroup$ @Ben, my hypothesis: SDS disrupts hydrophobic interactions as a surfactant. However, something like DNA has purely enthalpic interactions and will not undergo denaturation or be evenly coated by SDS. This is the underlying roots of my problems. $\endgroup$ – bobthejoe Jun 4 '12 at 19:18

The first step in troubleshooting is to run controls - run lanes with your protein input alone, and the conjugate alone (you may need to play with the type/percentage of the gel if the conjugate molecule is much smaller than your target protein - attaching biotin to a 50 kDa protein, for example) to see if the smear shows up. Depending on the type of conjugation you're using, the number of acceptor sites on the substrate, and the time and conditions of the conjugation reaction, you may be adding a highly variable number of conjugate molecules to your substrate. For example, when I first started working with adding fluorophores like Alexa and DyLight dyes to antibodies, the fluor/protein ratio was highly variable from reaction to reaction and between antibodies in the same reaction.

This is most likely what is happening in your case - the number of conjugate molecules being added to each substrate molecule is highly variable. If you haven't already, I'd suggest getting in touch with your conjugation kit vendor's tech support, as they likely encounter this problem all the time. You may need to change the type of conjugation chemistry, get a higher-quality conjugate, or purify the substrate before and/or after the reaction. One of the solutions to my antibody labeling problem I mentioned above was to purify the reaction products through a size-exclusion column. This had the dual effect of separating out the un-reacted antibody and fluorophore, and allowing us to pick different fractions of the conjugated product and decide which were best for our needs.

  • $\begingroup$ +1 Thanks! Issues with the suggestions so far. a) we're not conjugating a protein to another protein so standard commassie staining will not stain the conjugate molecule. b) we are the vendor since everything is done inhouse. c) While we can easily separate the reacted molecules from the unreacted, this doesn't answer the problem of quantifying the extent of the reaction. $\endgroup$ – bobthejoe Jun 4 '12 at 19:18

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