Western blot SDS-PAGE → transfer → detection
Separate proteins by size (SDS-PAGE), move them onto a membrane (transfer) and find one protein with antibodies. The skeleton rarely changes, but gel %, loading amount, antibody dilution and transfer conditions are adjusted for every experiment — use the "Variable explorer" below to see what each change does.
Key points
- Choosing a gel % that matches your protein size comes first
- Both target and loading control must stay below saturation to be quantified
- Optimal antibody dilution and blocking differ per antibody — compare 2–3 conditions side by side first
Materials
- Quantified protein lysate (e.g. BCA)
- 4× Laemmli sample buffer (with reducing agent)
- Polyacrylamide gel (hand-cast or precast)
- Running buffer (Tris-glycine-SDS)
- Transfer buffer (Tris-glycine + methanol)
- PVDF or nitrocellulose membrane, filter paper
- Blocking solution (5% non-fat milk or BSA in TBST)
- Primary antibody, HRP secondary antibody
- TBST (Tris-buffered saline + 0.1% Tween-20)
- ECL substrate, chemiluminescence imager
Steps
SDS-PAGE
- 💡 Membrane proteins can aggregate when boiled; 37–70 °C for 10–30 min is sometimes used instead.
- 💡 See the gel recipe under "Calculations" and "Variable explorer → Gel %".
Transfer
Antibodies · detection
Calculations
SDS-PAGE gel recipe
🔬 Variable explorer — what changes if…?
The skeleton stays the same, but concentrations, times and reagents change between experiments. Click an item to see what it affects and compare two conditions (A and B) side by side.
Gel percentage
What it isAcrylamide percentage sets the mesh size. A tighter mesh separates small proteins better.
Lower · lessLarge mesh: big proteins spread out well, but small ones run off with the dye front.
Higher · moreSmall proteins separate well, but large ones pile up near the top.
Typical choiceUsually 10–12%. Use a 4–20% gradient to see a wide size range at once.
Protein load (µg)
What it isTotal protein per lane — changes the signal of both target and loading control.
Lower · lessLow-abundance targets may not be visible.
Higher · moreBands smear and the abundant loading control saturates first, distorting normalisation.
Typical choiceUsually 10–40 µg of cell lysate. Run 5, 10, 20, 40 µg once to find the linear range.
Transfer time · methanol · membrane
What it isConditions for moving proteins from gel to membrane — the best choice depends on protein size.
Lower · lessShort or high methanol: large proteins (>150 kDa) stay in the gel.
Higher · moreLong: small proteins (<20 kDa) can pass through the membrane and be lost.
Typical choiceWet 100 V for 60–90 min, 20% methanol. For large proteins use ≤10% methanol and longer; for small ones shorter with a 0.2 µm membrane.
Blocking agent
What it isCovers empty sites on the membrane so antibodies do not stick everywhere.
Lower · lessToo short or dilute: blotchy, high background.
Higher · moreToo long or concentrated: can mask the target and weaken the signal.
Typical choice1 h at room temperature, 5%. Follow the antibody datasheet first.
Primary antibody dilution
What it isHow dilute the antibody is. More antibody raises both signal and background.
More concentratedToo concentrated (e.g. 1:250): more background and non-specific bands, and wasted antibody.
More diluteToo dilute (e.g. 1:20,000): the signal may be too weak to see.
Typical choiceStart from the datasheet dilution (often 1:500–1:2,000) and compare 2–3 around it.
Membrane (PVDF vs NC)
What it isMembrane material and pore size that hold the proteins.
Typical choicePVDF 0.45 µm in general; 0.2 µm for proteins below 20 kDa.
Exposure time · ECL
What it isHow long the chemiluminescent signal is collected.
Lower · lessShort: faint bands are missed.
Higher · moreLong: strong bands saturate (burn white) and differences can no longer be measured.
Typical choiceCapture several exposures and quantify one with no saturation warning.
Troubleshooting
| Problem | Common cause | Fix |
|---|---|---|
| No bands | Too little protein, failed transfer, antibody problem | Check transfer with Ponceau, load more, use more antibody, run a positive control |
| High overall background | Insufficient blocking, too much antibody, too little washing, dried membrane | Block longer, dilute antibody more, wash more, never let the membrane dry |
| Multiple bands | Non-specific binding, degradation, modifications such as phosphorylation or glycosylation | Dilute antibody, add protease inhibitors, check expected size and isoforms |
| Bands curve upward ("smiling") | Heat from high voltage, differing salt in samples | Lower voltage and keep cool, match sample buffer composition |
| White spots or blank patches | Bubbles between gel and membrane | Roll out bubbles when assembling the sandwich |
⚠ Safety Unpolymerised acrylamide is a neurotoxin — always wear gloves. Methanol is volatile and toxic; ventilate. Switch off the power supply before opening the tank.
This page summarises widely used general conditions. Reagent, kit and antibody manuals and your institution's safety rules take priority. Simulators marked "concept" are simplified models that show trends.
Analyse the results with free tools:
Western blot densitometry → BCA standard curve · loading volumes → Reagent & dilution calculator →