Electrophoresis (literally “to carry with electricity”) is a technique that uses an electric field to move charged molecules through a gel matrix.
For Western blotting, we use SDS-PAGE (Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis). Here’s what happens in simple terms:
SDS coats all proteins with a uniform negative charge
Heat and reducing agents unfold proteins into linear chains
Electric current pulls negatively charged proteins toward the positive electrode
Polyacrylamide gel acts like a sieve: small proteins move fast, large proteins move slow
The result? Proteins separate by molecular weight—smallest at the bottom, largest at the top.
One of the most brilliant features of SDS-PAGE is the two-layer gel system.
| Gel Layer | pH | Purpose |
|---|---|---|
| Stacking gel (top, 4–5%) | 6.8 | Concentrates all proteins into a tight band before they enter the resolving gel |
| Resolving gel (bottom, 6–20%) | 8.8 | Separates proteins by size |
Why two layers? Without a stacking gel, proteins would enter the resolving gel at different times, producing wide, blurry bands. The stacking gel uses the isotachophoresis effect to line up all proteins at the same starting line.
The stacking gel contains chloride ions (from Tris-HCl) that move quickly. Your protein samples contain glycine ions (from running buffer) that move slowly at pH 6.8. This creates a voltage gradient that sandwiches proteins into a thin zone—usually just 10–20 µm thick!
Polymerized SDS-PAGE gel (hand-cast or pre-cast)
Electrophoresis tank and lid with electrodes
Power supply
1X Running buffer (see recipe below)
Protein samples + ladder in loading buffer
Gel-loading tips
Remove the comb and any bottom tape (for pre-cast gels). Rinse wells with running buffer using a syringe—this removes unpolymerized acrylamide and straightens wells.
Place the gel cassette into the tank. For mini-gel tanks (like Bio-Rad Mini-PROTEAN):
Inner chamber: Fill with 1X running buffer (covers wells)
Outer chamber: Fill until buffer reaches the bottom of the gel
Critical check: No leaks from the inner chamber. If buffer drains out, your gel won’t run.
Thaw protein samples and ladder. Boil at 95°C for 5 minutes if not already denatured.
Using a gel-loading tip:
Insert the tip just below the top edge of the well
Expel slowly—don’t overfill
Load ladder in the first and last lanes (helps identify any smiling)
Loading guide (for 1.5 mm gel, 10 wells):
Ladder: 5–10 µL
Lysate: 10–30 µL (20–50 µg protein)
| Voltage | Speed | Best for | Risks |
|---|---|---|---|
| 80–100V | Slow (1.5–2 hours) | Sharp bands, high resolution | None |
| 120–150V | Medium (45–60 min) | Most routine blots | Minor heating |
| 180–200V | Fast (30–40 min) | Quick screening | Smiling, overheating |
My recommendation: Run at 100V until the dye front reaches the bottom of the stacking gel (about 20 minutes), then increase to 120–150V for the resolving gel. This gives sharp, straight bands.
Watch the dye front (bromophenol blue in your loading buffer). It runs ahead of all proteins and tells you where the smallest molecules are.
Stop the run when:
The dye front reaches the bottom of the gel, OR
Your smallest protein of interest is sufficiently separated
Don’t let the dye front run off the gel if you plan to stain with Coomassie—it carries small proteins with it.
Turn off the power supply. Remove the gel cassette. Carefully pry open the plates and cut off the stacking gel (discard it—it contains no target proteins worth keeping).
Your gel is now ready for transfer to a membrane.
| Component | Amount |
|---|---|
| Tris base | 30.3 g |
| Glycine | 144.0 g |
| SDS | 10.0 g |
| dH₂O | to 1 L |
To make 1X running buffer: Dilute 100 mL of 10X stock with 900 mL dH₂O.
pH check: Should be ~8.3–8.5. No adjustment needed if measured correctly.
Pro tip: Make fresh 1X running buffer for each run. Reusing buffer increases smearing and slows migration.
This section helps you troubleshoot intuitively.
| Factor | Effect |
|---|---|
| Molecular weight | Larger = slower (inverse relationship) |
| Gel percentage | Higher % = smaller pores = slower for all proteins |
| Voltage | Higher = faster (but more heat) |
| Buffer composition | Wrong pH or ionic strength = erratic migration |
Protein migration distance is logarithmically related to molecular weight. This means:
10 kDa vs 20 kDa → big separation
100 kDa vs 110 kDa → small separation
That’s why low-percentage gels are needed for large proteins—they barely move in high-percentage gels.
| Possible Cause | Fix |
|---|---|
| Gel percentage too high | Use lower % gel |
| Run stopped too early | Let dye front reach bottom |
| Voltage too low | Increase to 120–150V |
| Buffer too old | Make fresh 1X running buffer |
| Possible Cause | Fix |
|---|---|
| Uneven voltage across gel | Check that inner chamber isn’t leaking |
| Gel not polymerized evenly | Use fresh APS; mix thoroughly |
| Air bubbles under gel | Re-pour or tap bubbles out |
| Comb inserted crooked | Insert straight next time |
| Possible Cause | Fix |
|---|---|
| Missing glycerol in loading buffer | Add glycerol or use 2X loading buffer |
| Wells have unpolymerized acrylamide | Rinse wells with running buffer |
| Air bubble trapped | Use a longer, thinner loading tip |
Cause: The center of the gel gets hotter than the edges because heat dissipates poorly through the plastic cassette. Hotter gel = lower resistance = faster migration in the center.
Fixes:
Run at lower voltage (80–100V)
Pre-chill running buffer to 4°C
Run the tank in a cold room or ice bath
| Possible Cause | Fix |
|---|---|
| Too much protein loaded | Reduce loading amount |
| Sample not fully denatured | Boil longer; add fresh β-ME or DTT |
| Dirty gel or buffer | Use fresh gel and buffer |
| Gel overheated | Lower voltage or chill buffer |
| Possible Cause | Fix |
|---|---|
| No power to the tank | Check leads and power supply |
| Electrodes reversed | Red to red, black to black |
| No SDS in buffer | Add SDS to running buffer |
| Samples never loaded | Double-check before starting |
| Gel Type | Recommended Voltage | Approx. Time |
|---|---|---|
| Hand-cast, 10% | 100V (stacking) → 120V (resolving) | 60–75 min |
| Pre-cast, 4–20% gradient | 150V constant | 35–45 min |
| Large proteins (>150 kDa) | 80V overnight at 4°C | 12–16 hours |
| Small peptides (<10 kDa) | 200V (Tris-Tricine system) | 45–60 min |
Pre-run your gel – Run at 50V for 10 minutes before loading. This removes residual APS and ammonium persulfate, which can cause smearing.
Balance your lanes – Load equal volumes of loading buffer in empty lanes to prevent “edge effects” (distorted bands on the ends).
Mark your dye front – When the dye front reaches the bottom, note the time. That’s your run time for future consistency.
Keep buffer cold for high-voltage runs – Place the tank in an ice bucket or pre-chill buffer to 4°C.
Don’t touch the gel – Finger oils contain salts and proteases that ruin separation. Wear gloves and handle plates, not the gel.
Many beginners confuse these steps:
| Step | Purpose | Output |
|---|---|---|
| Electrophoresis | Separate proteins by size | Proteins in gel |
| Transfer | Move proteins from gel to membrane | Proteins on membrane |
You cannot skip electrophoresis and go straight to transfer—the proteins would all stick to the membrane in one messy blob.
Gel is fully polymerized (no liquid acrylamide smell)
Wells are rinsed with running buffer
Inner chamber is filled and not leaking
Outer chamber has enough buffer to cover the bottom
Lid is on correctly (red to red, black to black)
Power supply is set to the correct voltage (not current or power mode)
Timer is set
| Issue | Try to Fix? | Or Repour/Start Over? |
|---|---|---|
| Smiling bands | Lower voltage, chill buffer | No—finish the run |
| Leaking inner chamber | Add more buffer mid-run | No—but fix for next time |
| Bubbles in wells | Pop bubbles with a tip | Yes—bubbles ruin lanes |
| Gel cracked during pouring | No | Yes—cracked gel won’t separate evenly |
| Wrong ladder loaded | No (but note it) | Yes—you need size info |
Electrophoresis for Western blot isn’t magic—it’s physics and chemistry you can control. The perfect run comes down to three things:
Clean setup – No leaks, fresh buffer, rinsed wells
Right voltage – Slow enough for sharp bands, fast enough for your schedule
Cool conditions – Heat is the enemy of straight bands
Master these, and you’ll get publication-quality separation every time.
Now go run that gel—and don’t forget to watch the dye front.
Ahelixbio Pre-cast Protein Gel
Why: Excellent quality at lower price
Best for: Budget-conscious labs, high volume
Gradient: 4–20%
Price: ~$8.9/gel
