Hi all,
I`m trying to analyse PTMs (mainly phosphorylation) of a yeast protein by MS after enrichment of the tagged (e.g. TAP-tag) protein. I would like to perform the tryptic digestion on the magnetic beads, but so far I always have PEG-contamination in my digest despite washing the beads in ammonium bicarbonate. I heard that you could transfer the beads onto a filter and continue with a FASP-like procedure. Would anyone have a protocol for this? Other suggestions are also very welcome!
Thank you!
Hi Markus,
thanks a lot. That`s very helpful.
I actually also suspected at some point that the contamination might be coming from the beads, but never formally tested it.
I`m using M-270 Epoxy Dynabeads to couple the antibody to. Would you have a recommendation what might work better? Or would you be willing to share your information on streptavidin-beads? Then I could see if I can mage an analogous replacement for mine.
Thanks again.
I also met this problem. The solution is using Lammli Loading Buffer to boil the beads and run a SDS-PAGE for a very short time (stop when the sample just run into the seperating gel) and perform a In-Gel digestion.
Hi, thank you.
The original idea behind the on-bead digestion was to avoid incomplete elution. But the way you describe is the main one I used since and seems the least problematic.
Markus Hartl
Hi Markus, thanks a lot for the useful suggestion. We have similar problem of polymer background when using streptavidin C1 beads(Cat.No. 65002) . The Pierce streptavidin beads works fine in our hand regarding the polymer. However, we found there has a lot of non-specific signal in this beads compared with streptavidin C1 beads (See the silver stanning result in attacthed file). This comes two question:
1. What washing buffer should we used to reduce the background in Pierce beads (we have use very strong washing condition already)
2. We are planning using other beads for further testing. Could you tell us which beads you have test and that is not okay for on-bead digestion?
Would you give some suggestion in these issues, many thanks in advance.
Best,
Yuan
Markus Hartl
Hi Markus,
we use APEX2 method to perform proximity labelling in this experiment, that + and - indicated protein samples with or without biotin labelling.
Thanks for reminding that Pierce beads have the higher binding capacity. We use the same volume of beads, though the number of beads and endogenous biotinylated proteins could be part of the reason, the streptavidin-HRP signal in Pierce or C1 beads elution samples are similar with good specificity (+ vs -). Which means the beads are oversaturated in these samples, the differences between beads may mainly be caused by different coating material (as C1 beads are more hydrophilic). It's the truth that this is all about the signal to background ratio. So we will reduce the volume of the beads, and test different washing condition and also beads type in our system.
In terms of different bead types for on-bead digestion of biotinylated samples. The following method seems very useful and they use streptavidin beads from NEB. Article Protease‐resistant streptavidin for interaction proteomics
The wash buffer in the original BioID paper is harsh enough, thanks for all the useful information and we may try this recipe too.
Best,
Yuan
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