Sample Preparation for 2D Analysis

Sample Preparation

Samples should be supplied free of the following components that interfere with the first dimension isoelectric focusing step: salt, nucleic acids, polysaccharides, and lipids. Salt is a particular problem, as it leads to burning of IPGH strips. We recommend the following desalting methods:

  • Methanol precipitation
  • TCA/acetone precipitation
  • Spin columns e.g. Centricon

NOTE: If additional work needs to be done at the Mass Spectrometry Facility to remove contaminating components from samples then additional costs may be incurred.

Amount of Material

The following is a table giving a guideline to the amounts of protein required for various gel types: Analytical load (silver) mini gel 10-50ug total protein
Analytical load (silver) large gel 100-200ug total protein
Preparative load (coomassie) mini gel 100-500ug total protein
Preparative load (coomassie) large gel 1-3mg total proteinIf you are unsure of protein concentrations and need to know how much sample to send, the following is an approximate guide:

  • Bacteria are approx. half their dry-weight in protein
  • Plasma/Serum is approx. 60mg/ml protein
  • 1x107 Mammalian cells is approximately equal to 1mg of protein
  • Mammalian tissue (e.g. rat liver) requires a minimum of 10mg freeze dried material
  • Human Urine anywhere between 0.5mL to 10mL needed for a large analytical gel ("normal" control people will require 10mL)

Radioactivity

If you are sending radioactive samples, nature of radioactivity (including counts) must be stated with appropriate shielding to contain sample. Longer processing time may be necessary.

Pathogenicity

Biological samples coming into the Mass Spectrometry Facility should be accompanied by documentation of potential pathogenicity or pathogen free status. Otherwise the MS facility will presume all samples from human and animal origin are potential pathogens and will be treated accordingly.