I am using a biotinylated active site probe to label and isolate enzymes by electrophoresis and hopefully identification by mass spectrometry. But I get a lot of contaminants including streptavidin monomers and dimers when using streptavidin-coated magnetic beads even after using many methods of wash and elution. Unfortunately the streptavidin contaminants have the same molecular weight as our enzymes of interest. Any suggestions?
A couple of suggestions. First, can you do dual label of your active site probe and use a tandem affinity type system? This way you could first isolate with streptavidin, then clean up with binding to your second tag- getting rid of the streptavidin problem. Second, if you are using tandem mass spec you can identify multiple proteins in a mixture. We found that sometime just doing mass spec directly from our beads with a tandem affinity tagged protein (with the appropriate control samples in parallel) allowed us to find a number of interesting candidate interactions, confirmed with some previously known binding partners.
We are trying to identify the naturally occurring enzymes themselves so tandem affinity would not work. Doing on-bead digestion with controls is a possibility. Thanks for the suggestion.
Streptavidin is not glycosylated, if your proteins might be then you may be able to differentially stain them using PAS or lectins.
You could try streptavidin-agarose resins (e.g. from Pierce). I had less unspecific binding than with dynabeads.
Good to know. Thanks. I'll try the Pierce streptavidin agarose and if still contaminated with streptavidin, use lectin affinity to clean up.
Have you tried to preclear your sample with avidin beads before you do the biotinylation step. Briefly, add the avidin beads to your sample, remove the supernatent, then biotinylate your active site in the supernatent and pulldown with avidin beads. Also, higher cost beads (premium or high affinity) will contaminate less.
Yes, I do that because there are naturally occurring biotinylated proteins in the sample. Will look for the best grade of beads. Thanks.
We have used monomeric avidin magnetic beads from Bioclone (BcMag™ Monomer Avidin Magnetic Beads) with decent results to identify carbonylated proteins derivatized by biotin hydrazide. The elution takes place in mild conditions (2mM biotin) because the affinity of monomeric avidin for biotin is much less than that of streptavidin. So far we have not observed any contamination with avidin monomersand beads can be used a couple of times before losing some loading capacity. For more details you can browse the publications from Fred Regnier, for example. Hope this helps...
Use the Selective Reaction Monitoring method to look at fragment transitions. That way you can quantify each protein quantitatively.
Thanks for the tip on the Bioclone magnetic beads. I will definitely try them.
I use the protocol that you find in this paper to study carbonylation and it was good. Good luck!
We made very good experiences with the Streptavidin Sepharose High Performance from GE Healthcare. These beads have a very high binding capacity (6mg biot. BSA / ml resin) and you can use tiny amounts of resin (we use about 100 ul slurry per experiment) which helps you to reduce unspecific binding.
Thanks for the tip. That will help give a very concentrated sample.
Hi, 4 different types of streptavidin coated Dynabeads are available as well. They differ in size, binding capacity, and surface charge (all 4 are also available as a trial kit in order to find the right bead for the application).
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