Proteins submitted bound to beads
Please read the sections about the sources of detergent contamination, appropriate plastic and high quality solvents in the guidelines for proteins submitted in solution.
Here we provide a short summary of the most important points to consider when submitting proteins bound to beads, more details on how to properly wash the beads can be found in the protocols section (Note, that the protocols section is only accessible within the IMP/IMBA/GMI network).
Use the optimal bead to extract ratio
Please keep the volume of beads as small as possible. You can determine the optimal amount of beads and bead to extract ratio in a western blot before submitting your samples.
Washing of the beads and submission to MS
Following the immunoprecipitation transfer the beads to a new tube (preferentially Axygen maximum recovery tubes – available in the Protein Chemistry Facility). Wash your beads with your preferred wash protocol which has to be extended by several (5 to 10) MS-compatible wash steps using a detergent-FREE and protease inhibitor-FREE buffer (if possible, also Glycerol- and EDTA-free), such as 20mM Tris pH 8, 75 mM NaCl. After the last wash step remove the supernatant and submit the beads either frozen (magnetic beads) or at +5°C (non-magnetic Agarose beads) to our submission fridge/freezer.
Analyse a small aliquot by SDS-PAGE
Take a small aliquot (5-10%) of the beads during your last wash step and analyse it by both, silver staining and Western blot.
- For the silver-stained gel include a known amount of a protein ladder or different amounts of a BSA standard.
- For the western blot include your input lysate and a similar amount of the supernatant after the IP to be able to evaluate the success of the IP. In the optimal case, the bait protein shows a reduced signal in the supernatant but is not depleted from the lysate (see picture).
We can only accept your submission, if it contains an appropriately labelled picture of the silver-stained gel and Western blot.
In case you resubmit a similar experiment, we recommend controlling it again as described above, but we will accept the submission also with one of both pictures. In this case, please make a reference to the corresponding previous submission.