Western Blot Sample & Gel Loading Calculator

What is Western Blot Sample Loading?

Western blot sample loading involves preparing protein samples at consistent concentrations and volumes to achieve equal protein loading across all lanes of a gel. This consistency is crucial for:

• Accurate comparison of protein expression between samples
• Reliable quantification of band intensities
• Clean, interpretable results

Key Components

• Sample: Your protein lysate or extract at a known concentration (mg/mL)
• Loading Buffer: Contains SDS, reducing agent, glycerol, and tracking dye. Usually supplied as 2×, 4×, or 5× concentrate
• Diluent: Water or compatible buffer to bring the final volume up to the target lane volume

Understanding Units

For aqueous protein solutions, the unit conversion is straightforward:

This equivalence (since 1 mL = 1000 µL and 1 mg = 1000 µg) simplifies the calculation of sample volume needed to achieve a target protein mass.

Loading Buffer Concentration

Loading buffers are typically supplied as concentrated stocks (e.g., 4× or 5×) and must be diluted to 1× in the final sample:

For example, with a 4× stock and 20 µL lane volume: Buffer = 20 ÷ 4 = 5 µL

Typical Values

• Lane volume: 10–30 µL (depends on gel well capacity)
• Protein load: 10–50 µg total protein per lane (for cell lysates)
• Protein concentration: 1–10 mg/mL typical for cell lysates
• Loading buffer: 2×, 4×, or 5× stocks are common

When Target Load Cannot Be Achieved

If your sample concentration is too low relative to the target protein load, you may not be able to fit enough sample in the lane volume while leaving room for loading buffer. In this case:

• The calculator caps sample volume at the available space
• Achievable protein load will be less than the target
• Consider concentrating your sample before loading

Limitations

• Assumes accurate protein concentration measurement (BCA, Bradford, etc.)
• Does not account for sample viscosity or pipetting errors
• Does not specify heating conditions or reducing agents
• Cannot predict gel running or transfer conditions