Recommendations for Gel Band-based Protein Identification
- Use commercially available SDS-PAGE instead of home-made SDS-PAGE.
- Coomassie staining instead of silver staining if possible (contact the director if coomassie staining is not possible).
- Stain the gel in a closed container all the time to minimize contamination and destain the gel with milliQ water,
- Wear gloves the entire time of handling the samples.
- Take a digital image of the gel indicating the band(s) to cut and send the digital file to the director via email.
- 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.
Recommendations For in-solution (IP) based Protein Identification
- If possible, conjugate the primary antibody to solid support (preferably magnetic beads).
- Start with as much starting material as possible.
- Optimize antibody to antigen/bait protein ratio. Only use as much antibody as to be able to pull down >90% antigen/bait.
- Do not use a wash buffer containing carrier protein (e.g. BSA, milk).
- Verify the IP by Western Blot and submit the beads after the last wash without any liquid.
- 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.
- Store the beads at -20oC prior to submitting the sample.
Recommendations For Global Proteome and PTM analysis from Cells, Tissues and Bio-fluids
- 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.
- 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.
- 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.