SDS-PAGE Calculators

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SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is the standard laboratory method for separating proteins by molecular weight. SDS (a detergent) denatures proteins and coats them with negative charge proportional to their length, making all proteins migrate through the polyacrylamide gel matrix by size alone. Proteins are loaded alongside molecular weight markers (protein ladders), run through the gel under an electric field, and then stained or transferred to a membrane for further analysis (Western blot). SDS-PAGE is used for protein identification, purity assessment, molecular weight determination, and as the first step of Western blotting.

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How SDS-PAGE Works

Sample preparation: boil protein with SDS + reducing agent (β-ME or DTT) → SDS coats denatured protein with negative charge proportional to length (~1.4 g SDS per g protein); disulfide bonds are broken by reducing agent → all proteins migrate toward anode (+) based on size alone.

Gel Composition

Polyacrylamide gel: cross-linked polymer network; pore size is set by %T (total acrylamide) and %C (crosslinker bisacrylamide): higher %T → smaller pores → better resolution of smaller proteins. Typical: 8% for >80 kDa; 12% for 20–80 kDa; 15–18% for < 20 kDa. Gradient gels (4–20%) resolve a wide size range. Stacking gel: large pores, low pH concentrate proteins into thin bands. Resolving gel: smaller pores, high pH separates by size.

Staining Methods

  • Coomassie blue R-250 or G-250: Stains most proteins; detection limit ~0.1–0.5 μg/band; simple; not compatible with mass spectrometry
  • Silver stain: 50–100× more sensitive (1–5 ng/band); more variable; compatible with downstream analysis with special silver stain protocols
  • SYPRO Ruby: Fluorescent; linear dynamic range; compatible with mass spectrometry

MW Determination

Plot log(MW) of ladder bands vs. migration distance → linear regression → read MW of unknown from regression. Note: membrane proteins and highly charged proteins migrate anomalously (apparent MW ≠ true MW).

Glossary

SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel electrophoresis; separates proteins by molecular weight; SDS denatures and uniformly charges proteins; smaller proteins migrate faster through polyacrylamide.
Molecular Weight Ladder
A mixture of proteins of known MW run alongside samples; provides a log(MW) vs. migration calibration line for estimating unknown protein sizes from SDS-PAGE.
Western Blot
SDS-PAGE followed by protein transfer to a membrane and immunological detection with specific antibodies; identifies specific proteins by size and immunoreactivity.

Frequently Asked Questions

SDS (sodium dodecyl sulfate) is a detergent that denatures and coats proteins with uniform negative charge proportional to polypeptide length (~1.4 g SDS per g protein). Combined with heat and reducing agent (β-ME or DTT to break disulfide bonds), all proteins become rod-shaped SDS micelles with similar charge-to-mass ratios. Under an electric field, proteins migrate through the polyacrylamide gel matrix toward the positive electrode. Smaller proteins move faster through the pores; larger proteins move slower. Result: proteins are separated by molecular weight, from smallest (at bottom) to largest (at top).

Run a pre-stained or unstained molecular weight ladder alongside samples. After staining: (1) Measure migration distance from the stacking/resolving gel interface for each ladder band and for your protein band. (2) Plot log(MW) of ladder bands on y-axis vs. migration distance on x-axis. (3) Fit a linear regression (most labs use a semi-log plot). (4) Read off the MW of your protein from the regression line using its migration distance. Note: report this as 'apparent MW' because SDS-PAGE gives approximate molecular weight; anomalous migration occurs for membrane proteins (hydrophobic → less SDS binding), glycoproteins (carbohydrate doesn't bind SDS), and highly charged proteins.

SDS-PAGE separates proteins by size and allows visualization after staining. Western blot (immunoblot) adds an immunological detection step for specific protein identification: after SDS-PAGE, proteins are transferred ('blotted') from the gel to a nitrocellulose or PVDF membrane, then the membrane is probed with a primary antibody specific to the target protein, followed by a secondary antibody conjugated to HRP or fluorescent dye for detection. Western blot is more specific (detects one protein in a complex mixture) but requires an antibody; SDS-PAGE visualizes all proteins. Combination: SDS-PAGE confirms size; Western blot confirms identity.

%T (total acrylamide) determines pore size and resolution range: 6–8% gels: best for proteins > 80 kDa (histones, large enzymes, antibody chains). 10–12% gels: most versatile; proteins 20–80 kDa. 15–18% gels: small proteins < 20 kDa (insulin, histones, peptides). 4–20% gradient gels: broadest range; resolves 10–250 kDa simultaneously; popular for complex samples. For proteins you know are large (>150 kDa), low %T prevents them from running off the gel. For unknown proteins, 10–12% is a common default. Tricine-SDS-PAGE uses a different buffer system and resolves peptides < 5 kDa that standard glycine-SDS-PAGE cannot resolve.