isoelectric focusing
Summary
Isoelectric focusing is a gel electrophoresis method that separates proteins by their isoelectric point (pI) along a pH gradient. Each protein migrates until the surrounding pH equals its pI, where its net charge is zero and it stops. Separation depends on pI, not size.
Detail
Meaning: The gel holds a stable pH gradient, acidic at the anode (+) and basic at the cathode (−). A protein's net charge depends on the pH around it. Where pH is below its pI, the protein is net positive and moves toward the cathode, into higher pH. Where pH is above its pI, it is net negative and moves toward the anode, into lower pH. It stops where pH = pI, because net charge is zero and no force acts on it. If it drifts away, it regains charge and is pushed back, so it forms a sharp "focused" band.
Equation: pI = (pKa1 + pKa2)/2, where pKa1 and pKa2 are the two pKa values on either side of the neutral (zwitterion) form of an amino acid. Both are unitless. For an acidic or basic side chain, use the two pKa values that bracket the neutral form.
On the MCAT: A passage gives protein pI values and a gel pH range. You predict where each protein ends up or which way it moves at a stated pH. It is often paired with SDS-PAGE in two-dimensional gel electrophoresis, with isoelectric focusing first (by pI) and SDS-PAGE second (by mass).
Common confusion: Students treat it like SDS-PAGE and sort by size. Here, size does not set the final position, because the protein stops where its net charge reaches zero. Also, a protein below its pI is positive and moves toward the cathode, not the anode.
Sources
- Lehninger Principles of Biochemistry
- Biochemistry (Berg, Tymoczko, Gatto, Stryer)
- Voet's Biochemistry
Written for the MCAT and checked before it was published. For study reference only. Spotted an error? Let us know.