Sample Preparation Recommendations

Recommendations for Gel Band-based Protein Identification

  1. Use commercially available SDS-PAGE instead of home-made SDS-PAGE.
  2. Coomassie staining instead of silver staining if possible (contact the director if coomassie staining is not possible).
  3. Stain the gel in a closed container all the time to minimize contamination and destain the gel with milliQ water,
  4. Wear gloves the entire time of handling the samples.
  5. Take a digital image of the gel indicating the band(s) to cut and send the digital file to the director via email.
  6. Make an appointment to bring the gel over in a covered container with milliQ H2O.

Examples to run the gel for IP samples and Protein ID experiments.

Examples of gel bands

Recommendations For in-solution (IP) based Protein Identification

  1. If possible, conjugate the primary antibody to solid support (preferably magnetic beads).
  2. Start with as much starting material as possible.
  3. Optimize antibody to antigen/bait protein ratio. Only use as much antibody as to be able to pull down >90% antigen/bait.
  4. Do not use a wash buffer containing carrier protein (e.g. BSA, milk).
  5. Verify the IP by Western Blot and submit the beads after the last wash without any liquid.
  6. If non-MS compatible detergents are used in your buffers, wash IP with buffer not containing detergent and switch to new tubes a few times during washing.
  7. Store the beads at -20oC prior to submitting the sample.

Recommendations For Global Proteome and PTM analysis from Cells, Tissues and Bio-fluids

  1. For adherent cell keep the confluency 80-90%, after trypsinization, collect the cells by low speed centrifuge and wash the cells twice with ice cold phosphate-buffered saline(PBS), transfer into 1.5-ml tubes and centrifuge to remove supernatant completely. Bring the cell pellets on the dry ice or store the cell pellet at -800C until lysis.
  2. Suspension cell harvest: collect cells in 15-50 ml tubes, wash the cells twice with ice cold phosphate-buffered saline (PBS), transfer into 1.5-ml tubes and centrifuge to remove supernatant completely. Bring the cell pellets on the dry ice or store the cell pellet at -800C until lysis.
  3. Tissue collection: collect tissues in 1.5 ml tubes, and keep tissues in liquid nitrogen immediately after dissection and store at −80°C. For possible blood contamination, the samples should be rinsed twice with ice cold PBS.

Sample submission guidelines