The charge of a protein is the sum of contributions from its ionisable groups — the side chains of Asp, Glu, Cys, Tyr, His, Lys, Arg and the two terminal groups. Each behaves as a weak acid with its own pK:
Q(pH) = Σbasic ni · 10−pH / (10−pKi + 10−pH) − Σacidic nj · 10−pKj / (10−pKj + 10−pH)
The pI is the root of Q(pH) = 0. Hence the main rule: below its pI a protein is positively charged, above it negatively charged.
| Group | pK | Group | pK |
|---|---|---|---|
| Asp (D) | 4.05 | His (H) | 5.98 |
| Glu (E) | 4.45 | Lys (K) | 10.0 |
| Cys (C) | 9.0 | Arg (R) | 12.0 |
| Tyr (Y) | 10.0 | α-COOH | 3.55 |
| α-NH₃⁺ | 7.5 | Bjellqvist scale, as in ProtParam | |
A buffer works within a pKa ± 1 window, so you choose it for a particular working pH.
| Buffer | pKa, 25 °C | Working pH | ΔpKa / °C | Note |
|---|---|---|---|---|
| Sodium acetate | 4.76 | 3.6–5.6 | ≈ 0 | the acidic range |
| MES | 6.10 | 5.5–6.7 | −0.011 | does not chelate metals |
| Bis-Tris | 6.46 | 5.8–7.2 | −0.017 | |
| PIPES | 6.76 | 6.1–7.5 | −0.0085 | poorly soluble in the acid form |
| Sodium phosphate | 7.20 | 5.8–8.0 | −0.0028 | precipitates with Ca²⁺ and Mg²⁺ |
| MOPS | 7.20 | 6.5–7.9 | −0.011 | RNA gels |
| HEPES | 7.48 | 6.8–8.2 | −0.014 | compatible with magnesium |
| Tris | 8.06 | 7.0–9.0 | −0.028 | primary amine; strongly temperature-dependent |
Temperature. The pKa of Tris drops by 0.028 units per degree of warming, so on ice the pH rises: a buffer set to pH 8.0 at 25 °C will sit near 8.0 + 0.028 · 21 ≈ 8.6 in a 4 °C cold room. Set the pH at the temperature the buffer will work at.
Only Trp, Tyr and disulfides absorb at 280 nm (Pace et al., 1995):
ε₂₈₀ = 5500 · n(Trp) + 1490 · n(Tyr) + 125 · n(cystines) → c = A₂₈₀ / (ε · l)
Without tryptophans ε is small and the protein is nearly invisible on a NanoDrop — then you measure by Bradford. Nucleic-acid contamination shows up in A₂₆₀/A₂₈₀: ≈ 0.57 for pure protein, ≈ 2 for RNA.
| T7 promoter + lac operator | Works only in BL21(DE3); until IPTG is added the LacI repressor keeps the system silent. |
| His6 | Chelates Ni²⁺ — purification on Ni-NTA, elution with imidazole. |
| MBP | 40 kDa, sharply improves solubility and adds a second affinity on amylose. |
| TEV site | ENLYFQ↓S — the protease cuts here and releases the tags. |
| LIC | Ligation-independent cloning: T4 polymerase makes long single-stranded ends that simply anneal with the insert. |