Hello everyone,
I wanted to ask for some technical advice about BioID, in case anyone here is familiar with this technique. I'll try to keep it concise, but not too much.
I have created the clone with my protein of interest, ZNF148-BirA-FLAG, as well as a negative control to use as a “blank” for mass spectrometry, the Empty Vector-BirA-FLAG. I treated my cells with 50 µM biotin and induced them with 2 µg/ml doxycycline. I started performing BioID using streptavidin-sepharose beads, and by running a control with Western Blot, I could see that the BioID sample was highly enriched compared to the input when using an anti-FLAG antibody.
The problem arose when, during mass spectrometry analysis, my protein of interest (ZNF148) was also found sticking to the Empty Vector, albeit in much smaller quantities. Therefore, we decided to use magnetic streptavidin beads (Dynabeads™ M-280 Streptavidin, Thermo Fisher Scientific) to reduce non-specificity. However, with these magnetic beads, the BioID no longer works. In the Western Blot, the inputs are present, but where enrichment should occur, there is nothing.
I wanted to ask if anyone here has used these beads for BioID and if you'd be willing to share your protocol with me. Perhaps the washing steps or the lysis buffer I use for the sepharose beads are not suitable for the magnetic beads.
Below, I’ve included the ingredients of the lysis buffer, which I also use for the first four washing steps of the beads after performing BioID, as well as the other three washing buffers:
MOD RIPA BUFFER
- 1% NP-40
- 0.5% Na-deoxycholate
- 50 mM Tris-HCl pH 7.5
- 150 mM NaCl
- 1 mM EDTA
- 1 mM EGTA
- 0.1% SDS
WASHING BUFFER 1
- 2% SDS in H2O
WASHING BUFFER 2
- 50 mM Tris-HCl pH 7.5
- 12.5 mM NaCl
WASHING BUFFER 3
- 0.2% Na-deoxycholate
- 1% Triton X
- 500 mM NaCl
- 1 mM EDTA
- 50 mM Tris-HCl pH 7.5
Note:When using the sepharose beads, I didn’t perform the first four washes with the lysis buffer but with a buffer containing 50 mM NH4HCO3 pH 8 in H2O. Unfortunately, I couldn’t use it with the magnetic beads because they no longer attached to the magnetic rack, so I had to switch to the lysis buffer.
Thank you to anyone who can help!
This could be a simple carry-over problem in mass spec analysis instead of an experimental failure. If the only reason for switching from sepharose beads to dynabeads is the occurrence of small amounts in blank samples in mass spec analysis, it could be better to confirm the system first in terms of the abundant sample contamination (analyte transfer issue in LC)...
For magnetic bead application, sharing conditioning, binding, and elution buffer compositions could be beneficial other than the lysis and wash buffers. You may also lose the targets during the binding step or cannot elute from beads.
For the generic SPE development process, each buffer application should be subjected to analysis at the end of the attempt (western-mass spec-elisa-sds/page, etc) to find out where the targets are and to figure out the misapplication point.
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