A protein purification table is the scorecard of any enzyme isolation. It records, step by step, how much total protein and how much catalytic activity you carry forward, and from those two numbers it derives the metrics that tell you whether the purification is working: specific activity, yield, and fold purification.
How it works
For each fraction you provide three measured quantities, and the table computes the rest:
total protein (mg) = volume (mL) × protein conc (mg/mL)
total units = volume (mL) × activity (U/mL)
specific activity = total units / total protein (U/mg)
yield % = (step total units / crude total units) × 100
fold purification = step specific activity / crude specific activityThe first row you enter is treated as the crude starting material and becomes the 100 percent yield, 1.0-fold reference for everything below it.
Example purification walkthrough
Imagine purifying an enzyme from a bacterial lysate:
Step 1 — Crude extract: 100 mL at 10 mg/mL protein, 5 U/mL activity. Total protein: 1,000 mg. Total units: 500. Specific activity: 0.5 U/mg. Yield: 100%. Fold: 1.0.
Step 2 — Ammonium sulphate precipitation (40–60% cut): 20 mL at 8 mg/mL protein, 20 U/mL activity. Total protein: 160 mg. Total units: 400. Specific activity: 2.5 U/mg. Yield: 80%. Fold: 5.0.
Step 3 — Ion exchange chromatography: 10 mL at 0.5 mg/mL protein, 15 U/mL activity. Total protein: 5 mg. Total units: 150. Specific activity: 30 U/mg. Yield: 30%. Fold: 60.
Step 4 — Size exclusion chromatography: 5 mL at 0.1 mg/mL protein, 14 U/mL activity. Total protein: 0.5 mg. Total units: 70. Specific activity: 140 U/mg. Yield: 14%. Fold: 280.
The pattern is clear: yield steadily drops while fold purification rises. The 280-fold from crude to final fraction, at 14% yield, is a reasonable outcome for a four-step purification. The enzyme is now 280 times purer than it started.
Interpreting the fold and yield trade-off
Every purification step is a compromise between yield and purity. No step simultaneously increases both — each step removes some contaminating protein (good for fold) but also loses some of the target enzyme (bad for yield). How to interpret the pattern:
High fold, acceptable yield is the ideal outcome. A step that increases fold substantially while retaining 70%+ of activity is an efficient step worth keeping in the protocol.
High yield, minimal fold increase means a step is removing contaminating protein at roughly the same rate as it is losing target enzyme. This is worth re-optimising — adjust the gradient, fraction collection, or binding conditions to improve selectivity.
Low fold, low yield means a step is losing target enzyme faster than it is removing contaminants. This step is making the preparation worse and should be removed or redesigned.
Fold decrease (specific activity at a later step is lower than at an earlier step) indicates the enzyme is being inhibited or denatured at that step, or the activity assay is being interfered with by a compound being removed in that fraction.
Common purification steps and typical outcomes
| Step | Typical fold increase per step | Typical yield |
|---|---|---|
| Crude extract | 1× | 100% |
| Ammonium sulphate precipitation | 2–10× | 70–90% |
| Ion exchange chromatography | 10–100× | 50–80% |
| Affinity chromatography | 100–1000× | 60–90% |
| Size exclusion chromatography | 2–5× additional | 60–80% |
Affinity chromatography is the most powerful single step for enzymes where a suitable resin exists, because it exploits specific molecular recognition rather than bulk physical properties.
Tips and notes
Rows with zero protein are skipped for specific activity calculation to avoid dividing by zero. If your activity assay uses a non-linear or coupled reaction, confirm that the assay is in the linear range for each fraction — specific activity comparisons are only valid if the activity units are measured consistently across steps.