Q1: Why are some bands of the protein marker missing on the gel?
This usually relates to the gel concentration (percentage) and its effective separation range.Protein marker bands migrate differently depending on molecular weight and pore size:8–10% Tris-Glycine gels: Suitable for separating high-molecular-weight proteins, but small proteins (e.g., 10–15 kDa) may migrate too fast and even run off the gel.12.5–15% gels: Ideal for small proteins but may compress or trap large proteins (e.g., 180 kDa) near the top of the gel.
Q2: Extra bands appear in the protein marker lane — how to fix this?
Additional bands are usually caused by operational or storage issues:
vCross-contamination: Ensure samples do not leak between wells, especially in silver staining, which is highly sensitive.
vProtein degradation: Repeated freeze–thaw cycles or improper storage can degrade pre-stained/un-stained markers. Always use clean pipette tips and aliquot the marker immediately after opening to avoid contamination from multiple users.
Q3: What is the recommended loading volume for protein markers?
v0.75 mm and 1.0 mm gels: 3–5 μL
v1.5 mm gels: 8–10 μL
Q4: Why do pre-stained marker bands vary in width (broader on top, narrower below)?
Uneven band width typically results from excessive salt concentration, high loading volume, mismatched gel concentration, or overheating.Use fresh, pH-calibrated buffers, control sample salt concentration and loading volume, and avoid excessive heat during electrophoresis.
Q5: Why do marker bands gradually fade, leaving only the blue dye front?
This usually occurs due to repeated freeze–thaw cycles, prolonged room-temperature exposure, over-dilution, or boiling of non-boilable pre-stained markers.To prevent fading:Store aliquots at −20°C or −80°C as per instructions.Do not heat pre-stained markers if labeled “not heat-tolerant.”Use recommended dilution and reduction conditions.
Q6: What causes blurred or diffuse marker bands?
vReagents and gels: Use ultrapure water to prepare reagents; verify gel concentration and shelf life.Acrylamide/bis-acrylamide solutions (especially after opening) can lose effectiveness.
vElectrophoresis conditions: Avoid excessive voltage or long run times that cause overheating; pre-cool buffers if needed.
vBuffers: Use freshly prepared, pH-accurate electrophoresis buffers.
vSample storage: Store markers properly to prevent contamination and repeated freeze–thaw cycles.
Q7: Why are the bands curved (“smiling” or “frowning”)?
(1) Smiling bands (upward curve):Usually caused by overheating in the center of the gel.Solutions: Run electrophoresis at 4°C, increase the buffer volume in the outer chamber, or lower the running power.
(2) Frowning bands (downward curve):Caused by uneven electric fields or inconsistent gel thickness.
Check the following:
vNo air bubbles at the bottom of the gel.
vGel is fully polymerized.
vGlass plates are clean and residue-free.
(3) Other causes:
vEdge effects: Load markers in central lanes or include marker lanes on both edges.
vUneven gel casting or trapped air bubbles.
vUneven interface between stacking and resolving gels.
vLeaky electrophoresis tank leading to unstable voltage.
vExpired reagents such as acrylamide.
Aladdin: https://www.aladdinsci.com/