Virtual Protein Gel Electrophoresis

Protein Samples (one per line: Name: AMINO-ACID-SEQUENCE)

Example: BSA: MKWVTFISLLLLFSSAYSRGVFRRDTHKSEIAHRFKDLGEHFKGLVLIAFSQYLQQCPFDEHVKLVNELTEFAK Myoglobin: MGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMKASEDLKKHGATVLTALGGILK Lysozyme: MKALIVLGLVLLSVTVQGKVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGK

Gel Type

Protein Marker / Ladder

Acrylamide % 4 – 20

Voltage (V) 20 – 300

Current (mA)

Run Time (min) 10 – 300

Buffer System

Protein Stain

Reducing Agent (DTT / β-mercaptoethanol)

Temperature (°C) 4 – 40

Tip: Each Name: SEQUENCE line becomes one lane. Molecular weight is computed from the amino-acid composition. Migration is modelled using SDS-PAGE / Native-PAGE heuristics.

📑 On this page

Protein gel types — which to choose?
SDS-PAGE — principle & interpretation
Native-PAGE — what's different?
Tricine & gradient gels — small & wide-range proteins
Acrylamide %, voltage, buffer
Protein stains — sensitivity comparison
Protein markers / ladders
Reading your virtual gel
Troubleshooting & best practices

🧬 1. Protein Gel Types — Which to Choose?

Gel type	Denatures proteins?	Best for	MW range
SDS-PAGE	Yes (SDS + heat + reducing agent)	Molecular weight estimation, purity check	10–250 kDa
Native-PAGE	No	Enzyme activity, protein complexes	20–500 kDa
Tricine-SDS-PAGE	Yes	Small proteins & peptides	1–30 kDa
Gradient (4–20%)	Yes	Wide MW range in one lane	10–500 kDa
2D-PAGE	Yes	Proteomics — separates by pI then MW	10–200 kDa

Rule of thumb: For unknown samples, start with SDS-PAGE (denaturing). Use Native-PAGE only if you need to preserve activity or complexes.

📊 2. SDS-PAGE — Principle & Interpretation

SDS (sodium dodecyl sulfate) is an anionic detergent that:

Denatures proteins (unfolds them)
Binds uniformly at ~1.4 g SDS / g protein
Confers a constant negative charge-to-mass ratio

When combined with reducing agents (DTT or β-mercaptoethanol), disulfide bonds are broken. The result: all proteins migrate based only on their molecular weight.

Migration law

Relative mobility (Rf) ∝ −log(MW)

A plot of log(MW) vs migration distance is a straight line over the resolving range of the gel.

Reading the results

Observation	Interpretation
Single sharp band	Pure protein or single subunit.
Multiple bands	Subunits, isoforms, impurities, or proteolysis.
Band at expected MW	Correct protein.
Band at 2× expected MW	Possible dimer (disulfides not fully reduced).
Smear	Degradation, overload, or aggregation.
Band at dye front	Small peptide (< 10 kDa) — use Tricine gel.

🧪 3. Native-PAGE — What's Different?

Native-PAGE omits SDS and reducing agents, so proteins stay folded and active. Migration depends on:

Size (larger = slower)
Shape (globular vs fibrous)
Charge (depends on pH relative to pI)

Important: Native-PAGE does not give accurate molecular weights. Use SDS-PAGE for MW estimation.

When to use Native-PAGE

Detecting enzyme activity (zymogram)
Studying protein–protein complexes
Analysing oligomeric states
Preserving conformational epitopes

🔬 4. Tricine & Gradient Gels

Tricine-SDS-PAGE

Replaces glycine with tricine in the cathode buffer, allowing resolution of small proteins (1–30 kDa) that would run at the dye front on a standard Laemmli gel.

Use 10–16% acrylamide.
Recommended for peptides, small hormones, and cleavage products.
Schägger & von Jagow (1987) buffer system.

Gradient gels (4–20%)

A continuous gradient of acrylamide resolves a wide MW range in a single gel. The pore size decreases from top to bottom — small proteins keep migrating while large proteins are trapped at their pore limit.

Great for unknown samples.
Bands sharpen over time — best resolution in longer runs.
Commercially available as precast gels.

⚡ 5. Acrylamide %, Voltage, Buffer

Choosing acrylamide %

Acrylamide %	Resolvable MW range	Typical use
7.5%	50–500 kDa	Large proteins, complexes
10%	20–200 kDa	Standard SDS-PAGE
12%	10–100 kDa	Mid-range proteins
15%	10–50 kDa	Small proteins
16–20%	1–30 kDa	Peptides (Tricine)

Voltage

Stacking gel: low voltage (~80 V) to focus proteins into a sharp band.
Resolving gel: higher voltage (~120–150 V) for faster migration.
Too high (> 200 V) → heating, band distortion ("smiling").

Buffer systems

Buffer	pH	Best for
Tris-Glycine (Laemmli)	8.3	Standard SDS-PAGE
Tris-Tricine	8.3	Small proteins / peptides
Bis-Tris / MOPS	7.7	Neutral pH — better stability
Bis-Tris / MES	7.3	Small proteins, sharper bands
Tris-Acetate	7.0	Basic proteins, Native

💡 6. Protein Stains — Sensitivity Comparison

Stain	Colour	Sensitivity	Notes
Coomassie Brilliant Blue	Blue	~50 ng / band	Standard, cheap, reversible
Silver stain	Gray-black	~1 ng / band	Most sensitive; incompatible with MS
SYPRO Ruby	Red fluorescence	~1 ng / band	MS-compatible; broad linear range
Flamingo	Pink fluorescence	~1 ng / band	Very sensitive, low background
Ponceau S	Red	~100 ng / band	Reversible — used before immunoblot

Band intensity ≈ protein mass loaded. A band with 2× protein mass (at equal MW) will appear roughly twice as intense.

📏 7. Protein Markers / Ladders

Marker	MW range	Best for
Prestained broad range	10–250 kDa	General SDS-PAGE
Unprestained broad range	6.5–200 kDa	Accurate MW estimation
Low range	3.4–100 kDa	Small proteins
High range	40–500 kDa	Large proteins
Tricine marker	1.4–26.6 kDa	Peptides

Prestained vs unstained: Prestained ladders are visible during the run but less accurate for MW estimation. Unstained ladders give accurate MW but require staining after the run.

🔍 8. Reading Your Virtual Gel

The virtual gel generated by this tool shows:

Lane 1 — the protein ladder with MW labels on the right.
Lanes 2+ — your samples, one lane per input protein.
Stacking gel band — visible at the top (for SDS / Tricine gels).
Bright bands — larger proteins (higher mass → more stain).
Dim bands — small proteins or low-mass bands.
Migration arrow — direction of migration (top → bottom, toward +).

Estimating molecular weight

Identify your unknown band.
Note which ladder bands are immediately above and below it.
Estimate the position linearly on log(MW) scale — plot log(MW) vs distance for the ladder.
For high accuracy, use the reference ladder to build a standard curve.

Interpreting your results

Observation	Interpretation
Single sharp band at expected MW	Pure, correct protein.
Multiple bands	Subunits, isoforms, or impurities.
Band at ~2× expected MW	Dimer — reduce more thoroughly.
Broad smear	Degradation or heterogeneity (glycosylation).
Band near dye front	Very small protein — use Tricine gel.
No band visible	Too little protein or failed transfer.

🛠 9. Troubleshooting & Best Practices

Problem	Cause	Fix
Smiling bands (curved upward)	Overheating — too high voltage	Reduce voltage, use ice bath
Vertical streaks	Overloaded lane	Dilute sample
Diffuse bands	Old gel, degraded sample	Fresh gel, fresh sample
No bands	No protein, forgot stain, wrong pH	Check sample amount, re-stain
Multiple extra bands	Proteolysis, disulfide heterogeneity	Add protease inhibitors, reduce fully
Bands near top	Protein too large or aggregated	Lower acrylamide %, add urea
Poor resolution	Wrong gel %	Optimise acrylamide %
Bands smeared at bottom	Small proteins running too fast	Use Tricine gel

Safety: Acrylamide is a neurotoxin and carcinogen — always wear gloves, weigh in a fume hood, and dispose of waste properly. SDS is an irritant. Handle with care.

Pre-run checklist

✔ Acrylamide % matches target MW range
✔ Fresh APS and TEMED (polymerisation)
✔ Samples denatured with SDS + reducing agent (for SDS-PAGE)
✔ Ladder loaded in lane 1
✔ Buffer correct and fresh
✔ Electrodes connected (proteins migrate to +)
✔ Voltage ≤ 150 V for standard gels
✔ Stain chosen based on sensitivity need

🧬 Virtual Protein Gel Electrophoresis Simulator

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