2.1.1 Sample Loading

Experimental Procedures
Loading Samples
Add a sufficient amount of samples and molecular weight markers. SDS-PAGE separates proteins based on their electrophoretic mobility, which is a function of protein size and charge. We recommend loading 20-50 μg of total protein onto precast Tris-Glycine small-pore gels.

For high-molecular-weight targets, we suggest using Tris-Acetate gels and related buffers. A molecular weight marker should always be added to the lane adjacent to the test sample to serve as a reference point. Molecular weight markers (or ladder bands) are mixtures of purified proteins with known molecular weights. Using pre-stained markers allows visualization of the distance proteins migrate during electrophoresis, and subsequent confirmation of their electrotransfer to the membrane. Biotin-labeled markers enable visualization of ladder bands and antibody targets on the membrane. Adding both types of markers is highly useful, as they not only allow monitoring of the experimental progress but also facilitate result interpretation.

Key Notes for Sample Loading:

Use 20-50 μg of total protein or 100 ng of pure protein;
Try to keep the amount of protein in each lane the same, and the volume as consistent as possible;
Fill empty lanes with an equal volume of 1× loading buffer.