Explain what each step of SDS-PAGE and Western blotting does and how to judge a blot.
Why it matters
Gels for DNA tell you about genes. Many questions, though, are about proteins: is this protein made, how big is it, and does a treatment raise or lower it? SDS-PAGE separates proteins by size, and a Western blot then detects one specific protein with antibodies. Biochemists, biological technicians and some clinical labs use these methods, and knowing why each step exists is what lets you troubleshoot a bad blot.
SDS-PAGE: separating proteins by size
Proteins differ in shape and charge, so on their own they would not separate cleanly by size. SDS-PAGE fixes that:
- Sample preparation. Proteins are mixed with a sample buffer containing SDS (sodium dodecyl sulfate), a detergent that unfolds proteins and coats them with negative charge roughly in proportion to their length. A reducing agent (such as DTT or beta-mercaptoethanol) breaks disulfide bonds. Heating completes denaturation.
- Loading. Equal amounts of total protein (measured beforehand with an assay such as BCA or Bradford) go into each well, plus a pre-stained protein ladder of known molecular weights in kilodaltons (kDa).
- The gel. A polyacrylamide gel with a short stacking gel on top, which concentrates samples into sharp bands, and a resolving gel below, where separation happens. Higher acrylamide percentages resolve smaller proteins.
- Running. Proteins migrate toward the positive electrode; smaller ones move farther.
Afterwards you can stain the whole gel (for example with Coomassie) to see every protein, or move on to a Western blot. Unpolymerised acrylamide is a neurotoxin, which is one reason labs buy precast gels or train people before letting them pour gels.
Western blot: finding one protein
- Transfer. An electric current moves the proteins out of the gel onto a membrane (nitrocellulose or PVDF), keeping their positions. A reversible stain such as Ponceau S confirms the transfer worked.
- Blocking. The membrane is soaked in a protein solution (non-fat milk or BSA) so antibodies do not stick everywhere.
- Primary antibody. An antibody that recognises your target protein binds to it on the membrane.
- Washing. Buffer washes (often TBS or PBS with a little Tween detergent) remove unbound antibody.
- Secondary antibody. An antibody that recognises the primary antibody, linked to an enzyme (such as HRP) or a fluorescent dye. It amplifies and reports the signal.
- Washing again.
- Detection. For HRP, a chemiluminescent substrate produces light captured by an imager; fluorescent secondaries are imaged directly.
Reading a blot
- Size check. Is the band at the expected kDa, judged against the ladder? Modifications such as glycosylation or phosphorylation can shift apparent size.
- Loading control. A protein expected to be constant (for example beta-actin, GAPDH or a total-protein stain) shows whether lanes were loaded evenly. Compare the target band to it, never the raw band alone.
- Controls. A positive control lysate known to contain the protein, and ideally a negative control (for example a knockdown) that should lack it.
- Quantification. Densitometry gives band intensities. Results are semi-quantitative and only valid when the signal is not saturated.
Worked example. Target band intensity: control 12,000, treated 6,000. Loading control: control 20,000, treated 10,000. Normalised: control 0.60, treated 0.60. The apparent halving was just uneven loading: there is no real change.
Troubleshooting by step
| Symptom | Step to check first |
|---|---|
| No bands at all, ladder transferred | Primary antibody, target abundance, detection reagent |
| Ladder also missing from membrane | Transfer |
| Dark background everywhere | Blocking, washing, antibody too concentrated |
| Many extra bands | Antibody specificity, too much antibody, protein degradation |
| Bands smile or smear | Running conditions, overloading, sample prep |
Common mistakes
- Unequal protein loading, then interpreting band strength without a loading control.
- Saturated signal, which hides real differences.
- Skipping the transfer check.
- Reusing diluted antibody without tracking it.
- Over-editing blot images. Journals and quality systems require original, unedited images.
Quick recap
- SDS plus reducing agent plus heat: proteins separate by size on polyacrylamide.
- Western: transfer, block, primary, wash, secondary, wash, detect.
- Judge bands against the ladder, controls and a loading control.
- Troubleshoot by asking which step could cause the symptom.
Try it
The Do tab has a quiz on the order and purpose of each step.