Western Blot Calculator

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FREE TOOL

Calculate how much sample volume to load to get equal protein amounts in each lane.

Target Protein per Lane (µg)

Typical: 20–50 µg for most proteins.

Max Lane Volume (µL)

Mini gel wells: 15–25 µL.

Loading Buffer Stock

Number of Samples

Sample Concentrations (mg/mL) — one per line or comma-separated

Leave blank to calculate for a single concentration. Enter individual concentrations for mixed samples.

Calculate wet, semi-dry, or tank transfer buffer volumes for Western blot membrane transfer.

Transfer Method

Total Buffer Volume (mL)

Wet: 800–1000 mL. Semi-dry: 50–100 mL.

Methanol % in Transfer Buffer

SDS in Transfer Buffer

Calculate blocking solution and antibody dilution volumes for membrane incubation.

Membrane Area (cm²)

Mini gel blot ≈ 45 cm² (6×7.5 cm). Standard ≈ 80 cm².

Blocking Agent

Blocking Solution Volume (mL)

Antibody Incubation Volume (mL)

Primary Antibody Dilution (e.g. 1000 for 1:1000)

Secondary Antibody Dilution

Antibody Stock Concentration (mg/mL) — optional

If known, shows mass of antibody per volume.

🎞️ Western Blot Results

How to Use the Western Blot Calculator

Sample Loading tab: Enter target protein per lane (µg), max lane volume, loading buffer concentration, and individual sample concentrations. Get exact sample and loading buffer volumes per lane.
Transfer Buffer tab: Select wet or semi-dry transfer, total volume, methanol %, and SDS %. Get exact amounts of each component.
Blocking & Antibody tab: Enter membrane area, blocking volume, antibody volumes, and dilution factors to get exact antibody amounts to use.

About Western Blot Protein Loading

Equalising protein loading across lanes is critical for accurate Western blot comparison. The general approach is to measure protein concentration of all samples (using BCA, Bradford, or A280), then calculate how much volume of each sample contains the target amount of protein (e.g. 30 µg).

The loading buffer (Laemmli buffer) contains SDS, glycerol, bromophenol blue, and β-mercaptoethanol. It denatures the protein and gives it a uniform negative charge for SDS-PAGE separation. The final loading buffer concentration in the loaded sample should be 1× (e.g. add 5 µL of 4× buffer to 15 µL of sample).

If the calculated sample volume exceeds the lane volume, you need to concentrate the sample or reduce the target protein amount per lane.

Transfer Buffer — Methanol and SDS Guide

20% Methanol (standard): Good for most proteins under 100 kDa. Methanol removes SDS from proteins, improving binding to PVDF/nitrocellulose.
10% Methanol: Better for proteins above 100 kDa — less methanol allows larger proteins to transfer more efficiently.
0.1% SDS: Adding SDS improves transfer of high molecular weight proteins (>150 kDa) at the cost of slightly reduced membrane binding. Use with 10–20% methanol.
PVDF vs Nitrocellulose: PVDF requires activation with methanol before use. Nitrocellulose is simpler but more fragile.

Western Blot Calculator