🔬 Electrophoresis Simulator
Place amino acids in the gel and watch where they migrate
+
−
Anode (+)
Cathode (−)
origin
A
B
C
In the gel above, molecule B didn't move. Why?
Core Concepts
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What IS the Isoelectric Point (pI)?
The pH at which a molecule has no net charge — positive and negative charges are perfectly balanced.
Also called a zwitterion (German for "hybrid ion") — the molecule has charges, but they cancel out.
Example: 50 positive charges + 50 negative charges = net charge of zero
Also called a zwitterion (German for "hybrid ion") — the molecule has charges, but they cancel out.
Example: 50 positive charges + 50 negative charges = net charge of zero
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Electrophoresis Migration Rules
| Electrode | Charge | What Goes There | Remember |
|---|---|---|---|
| Anode | Positive (+) | Anions (negative molecules) migrate here | AN-ions go to the AN-ode AN-AN: Anions → Anode. Opposites attract — negative molecules go to the positive electrode. |
| Cathode | Negative (−) | Cations (positive molecules) migrate here | CAT-ions go to the CAT-hode |
At the isoelectric point → net charge = 0 → molecule doesn't migrate in either direction
The Formula
pI = (pKa1 + pKa2) / 2
The isoelectric point is the average of the two pKa values flanking the zwitterion
How to Read This
Most amino acids have one acid group (COOH, pKa < 7) and one base group (NH3+, pKa > 7).
The isoelectric point is halfway between these two pKa values — the pH where the positive charge from NH3+ exactly balances the negative charge from COO−.
On the exam: they may give you a titration curve and ask you to identify the pI. Look for the flat region between the two steep rises — that's where the zwitterion lives.
The isoelectric point is halfway between these two pKa values — the pH where the positive charge from NH3+ exactly balances the negative charge from COO−.
On the exam: they may give you a titration curve and ask you to identify the pI. Look for the flat region between the two steep rises — that's where the zwitterion lives.
Interactive Titration Curve
Tap any labeled point to learn what's happening at that pH
pKa1 (≈2.34) — This is the acid group (COOH) dissociating. At this pH, 50% of COOH groups have lost their proton (become COO⁻). The molecule is in its most cationic form: ⁺NH₃–CH₂–COOH is transitioning to ⁺NH₃–CH₂–COO⁻. Below this pH = fully protonated, positive charge dominates, migrates toward cathode.
pI (≈5.97) — The isoelectric point. Charges perfectly balanced: ⁺NH₃–CH₂–COO⁻. This is the zwitterion — one positive, one negative, net zero. Won't migrate in electrophoresis. This is always the flat region between two steep parts of the curve. pI = (pKa1 + pKa2) / 2 = (2.34 + 9.60) / 2 = 5.97
pKa2 (≈9.60) — This is the base group (NH₃⁺) losing its proton. At this pH, 50% of NH₃⁺ groups have become NH₂. Transitioning from ⁺NH₃–CH₂–COO⁻ to NH₂–CH₂–COO⁻. Above this pH = fully deprotonated, negative charge dominates, migrates toward anode.
What About Amino Acids with Extra Groups?
The Multi-Group Approach
Some amino acids have more than 2 ionizable groups (e.g., aspartate has 2 acid groups + 1 base group).
The Rule:
1. Start titrating from the side with the greater number of like groups
2. Titrate past the same number of groups as you see on the other side
3. The pI = average of the two pKa values you just titrated past
Example: Aspartate (2 acidic groups, 1 basic group)
→ Start from the acidic side (more groups there)
→ Titrate past 1 group (same as the number of basic groups)
→ pI = (pKa1 + pKa2) / 2 (the first two pKa values)
Example: 3 acidic, 2 basic
→ Start from acid side (3 > 2)
→ Titrate past 2 (matching the 2 basic groups)
→ pI = (pKa2 + pKa3) / 2
The Rule:
1. Start titrating from the side with the greater number of like groups
2. Titrate past the same number of groups as you see on the other side
3. The pI = average of the two pKa values you just titrated past
Example: Aspartate (2 acidic groups, 1 basic group)
→ Start from the acidic side (more groups there)
→ Titrate past 1 group (same as the number of basic groups)
→ pI = (pKa1 + pKa2) / 2 (the first two pKa values)
Example: 3 acidic, 2 basic
→ Start from acid side (3 > 2)
→ Titrate past 2 (matching the 2 basic groups)
→ pI = (pKa2 + pKa3) / 2
Clinical Correlations
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Gel Electrophoresis
A method for separating macromolecules based on size and charge.
Used in protein analysis, DNA fingerprinting, and diagnosing hemoglobin variants (like sickle cell).
Board application: knowing that acidic amino acids (Asp, Glu) migrate toward the anode (+) and basic amino acids (Lys, Arg) migrate toward the cathode (−).
Used in protein analysis, DNA fingerprinting, and diagnosing hemoglobin variants (like sickle cell).
Board application: knowing that acidic amino acids (Asp, Glu) migrate toward the anode (+) and basic amino acids (Lys, Arg) migrate toward the cathode (−).
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Titration
An experiment to determine the pKa of an unknown solution.
The half-equivalence point on the titration curve = the pKa (where 50% is dissociated).
The flat regions (buffer zones) are where the solution resists pH change — histidine (pKa 6.2) is the best amino acid buffer because its pKa is closest to physiologic pH (7.4).
The half-equivalence point on the titration curve = the pKa (where 50% is dissociated).
The flat regions (buffer zones) are where the solution resists pH change — histidine (pKa 6.2) is the best amino acid buffer because its pKa is closest to physiologic pH (7.4).
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