Assay standard curves guide

When this guide is more useful than the calculator alone

If you already know which curve model to run and only need the fitted concentration, go straight to the calculator. Stay on this guide when the harder question is whether the blank should be subtracted, whether the fitted shape is defensible, or whether your unknown should be treated as an extrapolation instead of a reportable result.

Protein assay vs ELISA: where the workflow splits

Protein assays such as BCA and Bradford often behave like narrower-range standard curves, so the practical choice is usually between a linear and a quadratic fit plus consistent blank handling. ELISA workflows are more often sigmoidal, so the real decision shifts toward 4PL vs 5PL, weighting, and whether the upper and lower plateaus are behaving sensibly.

• Open the protein standard curve calculator for BCA or Bradford standards, unknowns, and dilution-aware concentration estimates.
• Open the ELISA standard curve fitter for 4PL/5PL work, weighting trials, and back-calculated recovery checks.
• Use this guide to decide what those fitted outputs mean before you treat them as final.

Blank subtraction and baseline decisions

Blank subtraction is not an automatic improvement. Use it when the blank captures the same background signal that standards and unknowns carry, and keep the treatment consistent across the whole plate or run. If subtracting the blank changes the story too much, that is often a cue to inspect the assay setup rather than a reason to force the cleaner-looking answer.

For protein assays, pay attention to whether the zero standard really behaves like the unknown matrix. For ELISA, decide whether the 0 concentration row belongs in the fit, should be subtracted then excluded, or should stay outside the fit on a log-x workflow.

Choosing the fit model

Model choice should follow assay behavior, not preference for a higher-looking score. Linear and quadratic models can be appropriate for protein assays across modest ranges. 4PL and 5PL are better fits when the assay response is clearly sigmoidal and the plate spans upper and lower plateaus.

1. Start with the simplest model that matches the assay family.
2. Check residual patterns, not just one summary metric.
3. Only move to the more flexible model when it fixes a real mismatch rather than cosmetic noise.

Range checks, outliers, and extrapolation

Unknowns should ideally sit inside the bracketed standard range. If an unknown lands outside, the number becomes an extrapolation warning. That is often the right time to adjust dilution, rerun the sample, or tighten the standard range rather than accepting the first estimate as stable.

A single suspicious point also matters more than a smooth-looking headline metric. Outlier treatment should be explicit: say what was excluded, why, and whether the conclusion changes if that point stays in.

What to write in the report

A reproducible assay write-up needs more than the final concentration. Include the assay type, standard range, fit model, weighting if any, blank handling, excluded points, dilution factors, and whether any unknown was extrapolated. Those decisions explain why another person would trust your concentration estimate.

Recommended next pages

• Protein standard curve calculator (BCA / Bradford)
  Run the actual fit for BCA or Bradford standards, compare linear and quadratic models, and export unknown concentrations.
• ELISA standard curve fitter (4PL/5PL)
  Use this when your plate needs 4PL or 5PL fitting, weighting checks, recovery review, and unknown concentration estimates.
• A260 concentration & purity calculator
  Check nucleic-acid concentration and purity before the assay workflow when sample quality is still uncertain.
• Linear regression calculator
  Use a simpler regression page when you want a stripped-down look at fit behavior, slope, intercept, and residual interpretation.
• Biology calculators
  Return to the biology hub when you need the broader lab workflow around assay prep, qPCR, cell work, and molecular biology.

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