Technical Support FAQ

Print

Technical Support FAQ


Question:

The protocol to follow for Edman-based microsequencing after using the SYPRO Ruby protein blot stain and a protocol for preparing a sample for mass spectrometry after using the SYPRO Ruby protein gel stain.


Answer:

For accurate Edman-based microsequencing, you should transfer the proteins onto PVDF membrane, stain with SYPRO Ruby Protein Blot Stain (1703127) following the procedure in the instruction manual, and then partially destain the blot using the following procedure:

  1. Wash the blot by placing it face-down on a solution of 150 mM Tris, pH 8.8/20% methanol for 10 minutes with gentle agitation
  2. Rinse the blot four times for 1 minute each in dH2O
  3. Air dry the blot

Then you will be able to perform successful Edman sequencing.

Note: You cannot sequence proteins from gel slices -- this is not a viable application (the gel will clog the sequencer), but you can perform mass spec from gel slices.

For mass spec the following protocol can be used:

  1. After staining with SYPRO Ruby gel stain, excise the protein bands or spots from the gel
  2. Wash the gel slices 2x with 25 mM ammonium bicarbonate in 50% acetonitrile and 1x with 100% acetonitrile
  3. Dry the slices using a SpeedVac concentrator
  4. Add approximately 20 µl of a 20 mg/ml sequencing grade modified trypsin to the dried residue and incubate overnight at 37 degrees C
  5. Extract the peptides from the gel slices with 50% acetonitrile, 5% trifluoroacetic acid
  6. Dry the extract under vacuum
  7. Resuspend the peptides in 2 µl of 50% acetonitrile, 5% trifluoroacetic acid
  8. Spot 0.5 µl onto the plate of a MALDI-TOF mass spec
  9. Apply 0.5 µl of 10 mg/ml a-cyanocinnamic acid to the spot and air dry at room temperature prior to acquiring mass spectra
  10. Perform mass spectrometry


Get Help
Service and Support

Technical Support: 1-800-4-BIORAD (1-800-424-6723)

Bio-Rad ISO Certificates

Download Bio-Rad's ISO Certificates:

Sign Up for Bio-Rad Updates!
Enter your email address below to receive your choice of the latest news, promotions, and more.