I have used saturated phenol for protein extraction of plant leaf sample, but result show less protein spots in the 2-D Gel electrophoresis. Is the saturated phenol (pH=6.8) give less protein yield than Tris-equilibrated saturated phenol (pH=8.0). I want to know is pH is important or Tris-equilibration or both are important for protein extraction.
pH is important. Molecules like dna,RNA and protein are all charged entities. This charge will change with the pH of the solution. Charged molecules will tend to dissolve in the aqueous phase and non charged molecules will fractionate into the organic phenolic layer so if you change the pH until, say,DNA becomes uncharged then it will dissolve into the phenol layer but when it is charged it will stay in the aqueous layer. Because dna is more easily neutralised than rna it will move into the phenol as the buffering becomes more acidic. Those proteins carrying little charge will then have more dna contamination if you use acidic phenol instead of alkaline phenol
pH is important. Molecules like dna,RNA and protein are all charged entities. This charge will change with the pH of the solution. Charged molecules will tend to dissolve in the aqueous phase and non charged molecules will fractionate into the organic phenolic layer so if you change the pH until, say,DNA becomes uncharged then it will dissolve into the phenol layer but when it is charged it will stay in the aqueous layer. Because dna is more easily neutralised than rna it will move into the phenol as the buffering becomes more acidic. Those proteins carrying little charge will then have more dna contamination if you use acidic phenol instead of alkaline phenol
Obviously pH is important. The pH of the the solvent decides the net charge of a protein and the extent to which a particular protein can be solublized in aqueous medium or in non-aqueous medium. If a protein is neutralized (by charge) in a particular pH then it acts more like a non-polar molecule and tends to dissolve in a non-polar medium and not in the aqueous because only polar molecules tend to dissolve in a polar solvent like water. Also note that in my thesis work I had used 10% Water saturated phenol instead of Tris-Saturated Phenol. Water or Tris ; whatever is used to saturate the phenol is to bring the pH to a desired level for maximum protein solublization and extraction.
When you add water to an acid then think what might happen to the pH of the solution after you added enough water. Addition of water will shift the pH of the solution towards neutral pH. Similarly in case of Phenol which is solid at room temperature and is actually acidic in nature for using is diluted with water or with a suitable buffer and the pH checked afterwards is 6.6 to 6.8. The pH of the solution depends upon the concentration of Phenol in the solution. If further alterations are necessary then pH may be adjusted by using 0.1 N HCl or 1N NaOH or Tris base. Such ready-made solutions are already available in the stores. You can order directly for use. No need to prepare them. All you have to be concerned about is to stick to the pH and also know that the mixture is not actually a true solution as phenol and water/buffer are immiscible which is important for the phase separation.
Hope this helps.
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