Proteins can be separated on coated or uncoated capillary columns, and the choice of separation protocol depends on the specific properties of the target protein. The most important property is pl, which is determined by general gel electrophoresis or CE. Isoelectric spot focusing separation is a type of CE separation. Content from Compact Laboratory Guide to Molecular Biology (5th Edition)
Operation method
isoelectric point focusing
Principle
Isoelectric point aggregation (IEF) separation can be easily achieved by CE, which is also an effective first step in selecting the buffer system to be subsequently used for protein separation. Proteins and peptides are amphipathic, so their charge is determined by the surrounding carrier buffer. When a protein or peptide is in an electric field, it moves to a region where the surrounding pH is equal to its isoelectric point (pI). A pH gradient can be achieved in a packed capillary column by filling the column with a sample solution containing an amphoteric electrolyte. The high pH solution (sodium hydroxide) is placed in the cathode cell and the low pH solution (phosphoric acid) is placed in the anode cell. An electric field is passed through the column and the ampholytes and sample are moved to positions on the column corresponding to their respective pIs. If a molecule drifts out of the isoelectric point region, a charge is induced by the surrounding pH solution and the molecule then moves back to the 0 charge position. Narrow regions are formed by dissociation with pI in the order of 0.01 pH units. To determine the pI of a particular protein within the column, a marker with a known pI is added to the sample mixture, and the pI of the protein of interest is inferred between the markers. in the absence of an EOF, protein migration through the detector is required after separating the sample. Replacing the anode cell with a buffer in the cathode cell produces an EOF that allows the sample region to migrate through the detector. The separations discussed here assume that there is no EOF in the column during focusing, but separations can be performed in the presence of EOF.
Materials and Instruments
Sample containing 0.5 to 1.0 mg protein/ml dissolved in water. Move 1. Add the amphoteric electrolyte mixture to 0.5 ml of protein sample to achieve a final amphoteric electrolyte concentration of 2.5% (V/V). If desired, add IEF marker to achieve a final concentration of 0.1 mg/ml to calibrate the column. 2. Add the sample cell. Fill the capillary by pressurizing the cell (0.5 lb/in2 ). 3. Place 10 mmol/L phosphoric acid in the anode cell (negative) and 20 mmol/L NaOH in the cathode cell (positive). 4. 4. Focus the sample at a constant voltage of 8 to 10 kV for 4 to 6 minutes and detect the current until it stabilizes. 5. 5. Migrate the sample by placing 20 mmol/L NaOH at the anode, adjusting the voltage to 10 kV, and detecting the migration of the protein through the detector for 15-20 min. 6. 6. Wash the column with 10 mmol/L phosphoric acid at 0.5 lb/in2 for 1 min after each run. store the column in running buffer (short-term) or water (long-term) at room temperature. For more product details, please visit Aladdin Scientific website.
Amphoteric electrolytic mixture (pH 3~10) 20 mmol/L sodium hydroxide (stored at 4℃) 10 mmol/L phosphoric acid (stored at 4℃)
50 μm I.D. coated silicon capillary column CE apparatus