SDS-PAGE separates proteins by size and is the foundation of nearly every protein analysis workflow. Most issues with downstream techniques actually trace back to SDS-PAGE — so getting this step right pays off everywhere else.
Principle
Sodium dodecyl sulfate (SDS) coats proteins with a uniform negative charge proportional to their length. In an electric field through a polyacrylamide matrix, smaller proteins migrate faster, so you separate by molecular weight. A reducing agent (DTT or β-mercaptoethanol) breaks disulfide bonds.
Standard protocol
1. Prepare samples
Quantify protein with BCA. Mix 10–30 µg with 4× Laemmli buffer (SDS, glycerol, reducing agent, bromophenol blue, Tris-HCl). Heat at 95 °C for 5 minutes — except for membrane proteins (warm at 37–70 °C to avoid aggregation).
2. Choose gel percentage
| Target size | Gel % |
|---|---|
| >150 kDa | 6% |
| 50–150 kDa | 8–10% |
| 20–50 kDa | 10–12% |
| <20 kDa | 15% or gradient gel |
Gradient gels (4–20% or 4–15%) cover a wide size range in one lane and are a good default if you’re not sure of your target’s size.
3. Load and run
Load samples and a molecular weight ladder. Run at 80 V through stacking gel until samples enter the resolving gel, then 120–150 V. Stop when the dye front reaches the bottom.
4. Visualize or transfer
For protein detection, stain with Coomassie Brilliant Blue (~50 ng sensitivity) or silver stain (~1 ng). For western blot, proceed directly to membrane transfer.
Common problems and fixes
Smiling bands
Bands curve up at the edges. Caused by uneven heat — center gets hotter. Run at lower voltage, in a buffer-cooled apparatus, or in fresh running buffer.
Smeared lanes
Usually protein degradation or DNA contamination. Add fresh protease inhibitors during lysis, keep samples on ice, and consider DNase or sonication to shear genomic DNA.
Diffuse or fuzzy bands
Often overloading or buffer issues. Reduce sample amount, use fresh running buffer, don’t reuse buffer across runs.
No bands
Possibilities include too little protein, no reducing agent in sample buffer, or proteins running off the gel. Re-quantify, re-prepare buffer, time the run more carefully.
Bands at unexpected sizes
Could be PTMs, isoforms, dimers, or proteolytic fragments. Try denaturing more rigorously or longer reduction. Some heavily modified proteins simply don’t run at their predicted size.
Vertical streaks
Often well contamination from previous loading or precipitated samples. Spin samples briefly before loading and use clean tips for each lane.
Quality of life upgrades
- Pre-cast gels save time and improve consistency for routine work
- Stain-free technology lets you visualize total protein for normalization without Ponceau
- Use a pre-stained ladder to monitor migration during the run and during transfer
SDS-PAGE is forgiving when sample prep is good and unforgiving when it isn’t. Master the upstream steps — clean lysis, accurate quantification, fresh reducing agent — and the gel will reward you with sharp, even bands every time.
