Low concentrations of biological samples is one of the largest challenges encountered when attempting to structurally characterize biomolecules such as lipids, oligonucleotides, peptides and proteins. Therefore, any loss of these important biological molecules during isolation and analysis should be avoided. To this end, modification in sample handling protocols may be necessary.
Follow the KISS principle (keep it simple).
The following summarizes steps necessary to minimize sample losses:
- Attempt to eliminate or reduce the number of steps involving drying, lyophilization, and/or dialysis (It may be wise to totally eliminate dialysis as part of a purification procedure).
- When performing a series of chromatographic steps, reduce organic or salt concentrations by dilution before loading on to the next column.
- Arrange chromatographic separations intelligently, e.g., IEC, HIC, and/or SEC carried out prior to RPC.
- When necessary, utilize a cold acetone, methanol, ethanol or a chloroform/methanol/water precipitation method for optimal sample recovery.
- Keep all the solutions including the ones containing the waste (discarded material) until your experiment is over and you are satisfied with your data.
- Minimize sample exposure to new surfaces (pipette tips, vial etc.). Do not transfer concentrated samples from one vial to another.
- Avoid reducing HPLC fractions to complete dryness. For micro sequencing: Freeze fractions of interest using liquid nitrogen immediately after collection and store at -70° C until just prior to analysis.
- If buffer changes are necessary for other studies, attempt using volatile buffers such as ammonium formate or bicarbonate, and, if possible, avoid dialysis.
- Volatile buffers can be easily evaporated without compromising the sample. The sample will be dissolved in aqueous phase.
- Concentrated buffer stocks can be added to the sample for pH or buffer adjustments.
- Freeze sample-rich fractions at -70º C until analyzed and avoid multiple freeze/thaw cycles.