When running SDS-PAGE gels, a pre-stained protein ladder is more than just a colorful reference. It’s a visual tool that helps you estimate molecular weights, assess gel performance, and verify successful transfer in real time. In this guide, we’ll walk you through how to accurately read and interpret bands in a pre-stained protein marker ranging from 10–250 kDa.
🔍 What Each Band Represents
A pre-stained protein ladder contains a mixture of proteins with known molecular weights that are covalently linked to dyes. These bands run alongside your samples and serve as reference points on your gel and blot.
For example, NuSep’s Pre-Stained Marker (10–250 kDa) includes color-coded bands typically at:
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250 kDa
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150 kDa
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100 kDa
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75 kDa
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50 kDa
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37 kDa
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25 kDa
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20 kDa
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15 kDa
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10 kDa
Each band’s position on the gel reflects its relative size — with heavier proteins migrating more slowly (staying at the top), and lighter proteins moving farther down.
How to Use the Ladder for Molecular Weight Estimation
When running a Western blot or SDS-PAGE:
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Load your ladder in a separate lane, typically on the edge or in the middle of your gel.
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After the run, compare your sample bands to the ladder bands that are closest in size.
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Use the ladder’s known band sizes as a visual reference to estimate the molecular weight of your unknown proteins.
💡 Tip: Use imaging software or semi-log graphing if you need more precise estimation, especially between close bands.
Color Coding Makes a Difference
Many pre-stained markers use distinct dyes to color specific bands, helping you visually orient your results. For example:
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Blue: Most standard bands
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Red or Pink: Midpoint reference (usually 50 kDa)
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Green or Orange: Small band (e.g. 10 kDa)
These visual cues make it easier to quickly identify orientation and confirm your gel ran correctly.
Post-Blot Interpretation
After transfer, you can still see the ladder on the membrane, confirming a successful transfer and helping you align your blot image. This is especially helpful for imaging and publication — no extra staining needed.
Common Misinterpretation Pitfalls
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Assuming band intensity = protein quantity → Pre-stained bands may not be loaded equally.
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Ignoring gel distortions → Smiling gels or uneven loading can mislead placement.
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Using the ladder for quantification → It’s a size reference, not a concentration standard.
Summary
Understanding how to interpret your pre-stained protein ladder helps ensure your gel and blot are running as expected. From molecular weight estimation to blot orientation, it’s an essential tool for reliable, reproducible results.
Ready to use a clear, color-coded ladder in your next experiment?
👉 Try the 10–250 kDa Pre-Stained Marker from NuSep