SDS-PAGE Gel Calculator (Resolving & Stacking Polyacrylamide Recipes)
Generate bench casting recipes for discontinuous Laemmli SDS-PAGE gels. Select the optimal resolving gel percentage (6% to 18%) for your target protein molecular weight, scale across cassette thicknesses (0.75 mm to 1.5 mm), and calculate acrylamide, Tris, SDS, APS, and TEMED volumes.
Gel Geometry & Target Protein Size
Laemmli Bench Casting Recipes
SDS-PAGE Gel Calculator Principles: Polyacrylamide Gel Polymerization
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) separates proteins strictly on the basis of molecular mass. SDS is an anionic detergent that binds proteins at a constant ratio of ~1.4 g SDS per 1 g of protein (approx. 1 SDS molecule per 2 amino acid residues), masking intrinsic charges and conferring a uniform negative charge density and rod-like conformation.
Free Radical Polymerization
Ammonium persulfate (APS) decomposes to sulfate free radicals, accelerated by TEMED. Radicals initiate acrylamide monomer elongation crosslinked with N,N'-methylenebisacrylamide.
Pore Size & Sieve Effect
Higher acrylamide concentrations produce smaller average pore sizes. Low-MW proteins require 15–18% gels to avoid running off the bottom, whereas high-MW complexes require 6–8% gels.
Acrylamide Concentration vs. Protein MW Resolution Range
| Acrylamide (%) | Optimal MW Range | Representative Marker Proteins | Recommended Application |
|---|---|---|---|
| 6% – 7.5% | 50 – 250+ kDa | Myosin (200 kDa), β-galactosidase (116 kDa) | Large multi-subunit complexes, large enzymes |
| 8% – 10% | 25 – 150 kDa | Phosphorylase b (97 kDa), BSA (66 kDa), Ovalbumin (45 kDa) | General protein profiling, antibody heavy chain |
| 12% (Standard) | 15 – 80 kDa | BSA (66 kDa), Carbonic anhydrase (29 kDa), Trypsin inhibitor (20 kDa) | Standard recombinant expression, Western blotting |
| 15% | 10 – 50 kDa | Lysozyme (14.4 kDa), Aprotinin (6.5 kDa) | Small proteins, cytokines, cleavage tags |
| 18% | 4 – 30 kDa | Insulin chains (5.8 kDa), micro-peptides | Ultra-small peptides (Tris-Tricine recommended) |
Simulated Migration Pattern: Resolution Across Acrylamide Percentages
Higher percentage gels compress larger proteins near the top while spreading small polypeptides; lower percentages allow low-MW proteins to exit with the dye front but resolve massive proteins well.
The Laemmli Discontinuous Buffer System & Stacking Gel Mechanics
The brilliant sharpness of SDS-PAGE bands is created by isotachophoresis in the stacking gel:
1. Stacking Phase (pH 6.8)
Chloride ions (Cl⁻) act as leading ions with high mobility. Glycine is predominantly zwitterionic with near-zero mobility (trailing ion). Protein-SDS complexes have intermediate mobilities, getting compressed into an ultra-thin stack (< 0.1 mm) at the resolving gel boundary.
2. Resolving Phase (pH 8.8)
At pH 8.8, glycine fully ionizes into glycinate anions, increasing its mobility and overtaking the protein stack. Proteins then separate purely by molecular sieving through the polyacrylamide matrix.
SDS-PAGE Gel Calculator Worked Lab Example: Casting Two 1.0 mm 12% Gels
Scenario: Casting Two 1.0 mm 12% Mini-Gels (+10% Excess)
Target: Detecting Actin (42 kDa) and GAPDH (36 kDa). Total resolving volume = 11.0 mL; Stacking = 4.4 mL.
- Resolving Gel (12%): 3.63 mL H₂O + 4.40 mL 30% Acr/Bis + 2.75 mL 1.5 M Tris (pH 8.8) + 110 µL 10% SDS + 110 µL 10% APS + 11 µL TEMED.
- Stacking Gel (4%): 2.68 mL H₂O + 0.59 mL 30% Acr/Bis + 1.10 mL 0.5 M Tris (pH 6.8) + 44 µL 10% SDS + 44 µL 10% APS + 6.6 µL TEMED.
Frequently Asked Questions (FAQ)
How do I choose the right resolving gel percentage for my protein?
General guidelines: 6% for 50–250 kDa; 8% for 30–180 kDa; 10% for 20–100 kDa; 12% for 12–70 kDa (best for housekeeping markers); 15% for 8–45 kDa; 18% for small peptides (< 30 kDa).
Why does the resolving gel require an alcohol or water overlay during polymerization?
Atmospheric oxygen terminates acrylamide free radical polymerization. Overlaying with isopropanol or water creates an oxygen barrier and establishes a flat, level interface for subsequent stacking gel casting.
Why must APS (ammonium persulfate) be prepared freshly?
APS degrades rapidly in aqueous solution via spontaneous hydrolysis. Degraded APS fails to generate sufficient sulfate radicals, leading to incomplete or sluggish polymerization and runny gels.
What is the role of TEMED in gel casting?
TEMED is a tertiary aliphatic amine that accelerates the homolytic cleavage of persulfate into sulfate free radicals. Always add TEMED immediately before pouring as polymerization begins within minutes.
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