SDS-PAGE and Western 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? SDSSodium dodecyl sulfate, a detergent that unfolds proteins and coats them with negative charge roughly in proportion to their length.-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:

1. 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.
2. 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).
3. 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.
4. 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

1. 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.
2. BlockingSoaking the membrane in a protein solution such as milk or BSA so antibodies do not stick everywhere.. The membrane is soaked in a protein solution (non-fat milk or BSA) so antibodies do not stick everywhere.
3. Primary antibodyAn antibody that recognises and binds the target protein on the membrane.. An antibody that recognises your target protein binds to it on the membrane.
4. Washing. Buffer washes (often TBS or PBS with a little Tween detergent) remove unbound antibody.
5. Secondary antibodyAn antibody that recognises the primary antibody and carries an enzyme or dye that reports the signal.. An antibody that recognises the primary antibody, linked to an enzyme (such as HRP) or a fluorescent dye. It amplifies and reports the signal.
6. Washing again.
7. 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 controlA protein expected to be constant, used to check that lanes were loaded evenly before comparing target bands.. 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. DensitometryMeasuring band intensities from a blot image; semi-quantitative and only valid when the signal is not saturated. 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

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.