Ampholytes removal from proteins

With carrier ampholytes, concentrations of about 2% (w/v) are best. Ampholyte concentrations below 1% (w/v) often result in unstable pH gradients. At concentrations above 3% (w/v), ampholytes are difficult to remove from gels and can interfere with protein staining. 

V. PREPARATIVE ISOELECTRIC FOCUSING A. Removal of Ampholytes from Proteins... 

The proper choice of ampholyte range is very important to the success of an IEF fractionation. Ideally, the pH range covered by the focused carrier ampholytes should be centered on the pis of the proteins of interest. This insures that the proteins of interest focus in the linear part of the gradient with many extraneous proteins excluded from the separation zone. Carrier ampholyte concentrations of about 2% (w/v) are best. Concentrations of ampholytes below 1% (w/v) often result in unstable pH gradients. At concentrations above 3% (w/v) ampholytes are difficult to remove from gels and can interfere with protein staining. 

Removal of Carrier Ampholytes from Protein Fractions... 

The carrier ampholytes have comparatively low molecular weights. Thus they can be removed from the proteins of the sample by dialysis or by using a molecular sieve such as Sephadex gel. 

Each stored sample is then loaded on a reversed-phase protein trap column attached to a second six-port valve located on the capillary LC system. Proteins and peptides in the stored sample are captured on the trap column and washed with a solution of water-acetonitrile-fomic acid (94.9 5.0 0.1) at a flow-rate of 20pLmin for 3 min. Most of the ampholyte, urea and DTT are removed during this washing step. The protein fraction is then eluted from the trap column with mobile phase and further separated on a C4 reversed-phase column (5 cm X 300 pm i.d.). Mobile phase A [water-acetonitrile-fomic acid (94.9 5.0 0.1)] and mobile phase B [acetonitrile-water-acetic acid (94.9 5.0 0.1)] are delivered at a flow-rate of lOpLmin using a two-step gradient of 40min (phase B from 20 to 60%) and 2 min (phase B from 60 to 90%). The sample eluted from the RPLC column is sent directly into the ESI-QTOF mass spectrometer. 

Thus the surface charges of proteins, and of other ampholytes, may be explained by the addition and removal of hydrogen ions, i, e., protons. Charges on surfaces are frequently said to arise from "preferential adsorbtion of ions of one charge. This is, however, inadequate as explanation as long as the reason for the adsorbtion remains in doubt. 

Electrophoresis is then done, perpendicular to the direction of electrofocusing. The main advantage of this perpendicular treatment is the rapid removal of the carrier ampholytes from the protein zones. The... 

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