Protein Labeling Calculator
Protein Labeling Calculator
Accurately calculate the amount of labeling reagent (NHS-ester dyes, biotin, fluorescent tags, etc.) needed for your protein based on established bioconjugation chemistry.
µg ( nmol)
Final concentration of reagent in reaction: µM
Expected Degree of Labeling (DOL/DPR): ≈
Always verify final DOL by absorbance (Protein & Dye method) after purification.
About the Protein Labeling Calculator
The Protein Labeling Calculator is an essential online tool designed for researchers, biochemists, and biotechnologists who perform covalent protein labeling using NHS-ester chemistry (the most common method for attaching fluorescent dyes, biotin, PEG, or other functional tags to proteins).
This free Protein Labeling Calculator helps you determine the exact amount of labeling reagent needed to achieve your desired dye-to-protein ratio (D/P or Degree of Labeling, DOL) while avoiding over- or under-labeling that can impair protein function or reduce signal quality.
Why Protein Labeling is Important
Protein labeling is a cornerstone technique in modern life sciences. Fluorescently labeled proteins are used in:
- Fluorescence microscopy & live-cell imaging
- Flow cytometry & cell sorting
- FRET and protein–protein interaction studies
- Western blotting & ELISA enhancement
- In vivo imaging and pharmacokinetics
- Proteomics and affinity purification
Precise control of the degree of labeling is critical: too few dyes result in weak signals, while excessive labeling can alter protein conformation, reduce solubility, or quench fluorescence.
Scientific Basis & Formulas Used in This Calculator
All calculations follow peer-reviewed bioconjugation protocols (Thermo Fisher, Sigma-Aldrich, and publications in Bioconjugate Chemistry, Nat. Protocols, etc.).
Step-by-step calculation:
- Protein amount (nmol) = (Concentration × Volume × 1000) / MW
- Desired label molecules = Protein amount × Desired D/P ratio
- Reagent needed (nmol) = Desired label molecules × Excess fold
- Mass of reagent (µg) = Reagent nmol × Reagent MW / 1000
- Reagent concentration in reaction = (Reagent nmol / Volume) × 1000 µM
When & Why You Should Use This Protein Labeling Calculator
Use this calculator whenever you are labeling antibodies, enzymes, serum albumins, streptavidin, or any protein with amine-reactive probes (NHS, TFP, or sulfo-NHS esters). It eliminates guesswork and reduces costly reagent waste.
Common dyes this calculator supports: Alexa Fluor® series, Cy® dyes, FITC, Rhodamine, DyLight®, Atto dyes, biotin-NHS, PEG-NHS, and many more.
User Guidelines & Best Practices
- Protein should be in amine-free buffer (e.g., PBS, HEPES, bicarbonate pH 8.0–8.5)
- Recommended protein concentration: 2–10 mg/mL
- Typical excess: 5–15 fold for fluorescent dyes, 10–20 fold for biotin
- Reaction time: 30–60 min at room temperature or overnight at 4 °C
- Remove excess dye using size-exclusion spin columns or dialysis
- Always measure final DOL spectrophotometrically using the formula:
DOL = (A_dye × ε_protein) / ((A_280 – CF × A_dye) × ε_dye)
Purpose of This Free Tool
Our mission at Agri Care Hub is to provide scientists with accurate, free, and easy-to-use bioinformatics and bioconjugation tools. This Protein Labeling Calculator is part of that commitment.
For in-depth technical reviews on protein labeling methods, we recommend the excellent resource at Sino Biological – Protein Labeling.
Frequently Asked Questions
What is a good starting Dye-to-Protein ratio?
For most imaging applications, 2–4 dyes per IgG and 4–8 dyes per streptavidin give optimal brightness without loss of function.
Why do we add excess reagent?
NHS-esters hydrolyze in aqueous buffer (half-life ~20–60 min at pH 8). Excess ensures sufficient active ester remains to react with lysines.
Can I use this for click-chemistry or maleimide labeling?
This calculator is optimized for NHS-ester chemistry. Maleimide (thiol) labeling requires different molar ratios (typically 5–20× excess).
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