Why Should Samples Not Be Loaded on Both Sides When Determining Protein Molecular Weight by SDS-PAGE
SDS-PAGE is a widely used technique for protein separation and molecular weight determination. In this method, sodium dodecyl sulfate (SDS) binds to proteins, conferring a uniform negative charge, thereby allowing migration toward the anode under an applied electric field. However, the distribution of the electric field in the electrophoresis buffer can be influenced by sample loading positions.
When samples are loaded on both sides of the gel, local ion concentration in the buffer is altered, leading to an uneven electric field distribution. This non-uniform electric field can induce unintended ionic movement around the sample wells, creating an effect akin to electroosmotic flow or field distortion, which disrupts the expected migration path of proteins. This phenomenon results in abnormal protein band broadening and reduced separation resolution, particularly affecting proteins of lower molecular weight due to their higher susceptibility to minor electric field variations.
To mitigate this effect, it is recommended to avoid loading samples on both edges of the gel. This practice helps maintain a stable electric field, minimizes unintended ionic movement, and enhances the accuracy of molecular weight determination as well as overall separation efficiency.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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