Antibody Working Dilution Calculator: µg/mL and 1:n Titer
Plan primary or secondary antibody working solutions from either a target mass concentration (such as 2 µg/mL from a 1 mg/mL stock) or a declared 1:n datasheet dilution ratio. The interactive workspace loads a checked 10 mL dilution example and flags when an uncalibrated ratio lacks a stock concentration basis.
Loads the independently checked preparation fixture where a 1 mg/mL stock is diluted to 2 µg/mL in 10 mL final blocking buffer (requiring 20 µL stock and 9.98 mL diluent, equivalent to a 1:500 dilution factor).
Connect volumetric 1:n ratios to actual protein concentration
Immunodetection protocols for western blotting, immunofluorescence, immunohistochemistry and flow cytometry frequently mix two reporting conventions: mass concentration in micrograms per millilitre and volumetric ratios such as 1:500 or 1:2,000. When the stock concentration is 1.0 mg/mL (1,000 µg/mL), dividing 1,000 by the dilution factor gives the working concentration in µg/mL directly. When a vendor ships a lot at 0.5 mg/mL or 1.8 mg/mL, however, copying a historical 1:1,000 volume ratio silently changes the active antibody concentration.
The workspace lets you enter either the target concentration or the dilution ratio while keeping the stock concentration visible. If no stock concentration is available, the tool computes the transfer volumes and attaches an explicit flag noting that absolute concentration is unrecorded.
Track clone, lot and minimum pipette transfer limits
Always record the antibody clone name, catalogue number, lot number and target assay alongside the dilution calculation. Check the required stock draw against your pipette minimum-transfer threshold so that viscous glycerol-containing stocks are not drawn at sub-microlitre volumes.
Equations and variables
Dilution factor from mass or molar concentrations
F = cstock / ctarget; Vstock = Vtotal / F
- F
- Dimensionless dilution factor (greater than or equal to 1)
- cstock, ctarget
- Stock and working concentrations converted to a common mass or molar unit
- Vtotal
- Total batch volume across all vessels (N × Vvessel) (mL)
- Vstock
- Required volume of concentrated antibody stock (mL)
Diluent balance for blocking or staining buffer
Vdiluent = Vtotal − Vstock
- Vdiluent
- Volume of blocking buffer or assay diluent required (mL)
- Vtotal
- Total working solution volume prepared (mL)
- Vstock
- Volume of antibody stock added (mL)
Scope and limitations
- A 1:n ratio (such as 1:1,000) defines a volumetric transfer fraction but does not establish the final µg/mL concentration unless the lot-specific stock concentration is known and entered.
- Converting between mass concentration (mg/mL) and molarity (nM) requires the molecular weight of the specific immunoglobulin class or conjugate, which is not assumed automatically.
- Arithmetic dilution planning does not predict epitope accessibility, non-specific background or optimal titration for a new tissue or cell line.
Sources and provenance
- Calculator inputs and independent arithmetic verificationOriginal synthetic inputs; see individual fixture notices
- Independent antibody-dilution verification casesPython fractions and decimal exact arithmetic reference
Technical questions
Why is reporting final antibody concentration in µg/mL preferable to quoting only a 1:500 or 1:1,000 ratio?
Purified monoclonal antibody lots often vary in stock concentration from 0.2 mg/mL to 2.0 mg/mL across vendors or even between affinity-purified lots. A 1:500 dilution of a 1.0 mg/mL stock yields 2.0 µg/mL, whereas a 1:500 dilution of a 0.2 mg/mL lot yields only 0.4 µg/mL. Recording both stock concentration and final µg/mL keeps the assay reproducible across lots.
When is a 1:n ratio used without a stock mass concentration?
Unpurified antisera, hybridoma culture supernatants and ascites fluids often lack an isolated antigen-specific immunoglobulin mass concentration. In those cases, the volumetric dilution factor is the primary experimental quantity and should be paired with the exact lot identifier.
What if a high dilution factor requires less than 1 µL of antibody stock?
Preparing 1 mL of a 1:10,000 secondary antibody solution requires only 0.1 µL of stock, which cannot be pipetted accurately with standard air-displacement micropipettes. Either scale up the working volume if buffer is inexpensive or prepare a two-step intermediate dilution.
Connected workflows
- 4PL standard-curve interpolation
Interpolate antigen unknowns after running an immunoassay standard curve.
- Protein A280 concentration calculation
Measure purified immunoglobulin stock concentration by UV absorbance at 280 nm.
- HER2 and TROP2 ADC trial-record exploration
Explore clinical antibody–drug conjugate trial records in the pipeline workspace.