Hi Shaima,

GAPDH may migrate differently depending on the gel buffer system.

• Tris-Glycine (8-16%): Can cause differences in charge/mobility, leading to apparent double bands. Tris-Acetate (3-8%): Might provide conditions where the protein migrates as a single form.

Hope it helps.

A.Baki

The abnormal migration of housekeeping proteins (such as GAPDH) may be related to the pH of the buffer system, ionic strength, or gel pore size. If double bands are observed, the following verification methods are recommended: ✅ Repeat the experiment using the same batch of lysis buffer; ✅ Switch to alternative housekeeping proteins (e.g., β-actin, α-tubulin); ✅ Optimize electrophoresis conditions (e.g., reduce voltage, extend electrophoresis duration).

The 8-16% Tris-glycine gels might have higher resolution at certain protein sizes, which can sometimes reveal post-translational modifications or protein isoforms (e.g., different isoforms of GAPDH, phosphorylated forms, etc.), causing double bands to appear. The 3-8% Tris-acetate gels might not resolve these modifications as clearly, resulting in a single band. Try using a narrower range of Tris-glycine gels, such as a more specific 10-12% gel, to see if that resolves the double bands or results in more consistent banding like the 3-8% gels.

Check for isoforms or PTMs - Confirm that the double bands are indeed GAPDH isoforms or modified versions of GAPDH by including appropriate controls (e.g., knocking down GAPDH or using a different GAPDH antibody).