The SDS-PAGE Gel Calculator takes the guesswork out of gel preparation by computing exact volumes of every component — acrylamide, Tris buffer, SDS, APS, and TEMED — for both the resolving and stacking gels. Used daily by researchers, students, and lab technicians, it supports any acrylamide percentage from 6% to 20% and scales recipes for multiple gels simultaneously.
Typical range: 8–18%
Total volume for resolving gel.
Typically 2–4 mL for mini gels.
For a 12% resolving gel at 10 mL total volume, using 30% acrylamide stock and 10% APS:
Acrylamide = (12 ÷ 30) × 10 mL = 4.000 mL
1.5 M Tris-HCl pH 8.8 = 10 mL × 0.25 = 2.500 mL
10% SDS = 10 mL × 0.01 = 0.100 mL
10% APS = 10 mL × 0.01 = 0.100 mL
TEMED = 10 mL × 0.001 = 0.010 mL
Water = 10 − 4.000 − 2.500 − 0.100 − 0.100 − 0.010 = 3.290 mL
Mix water, acrylamide, Tris, and SDS first, then add APS and TEMED last and pour immediately.
| Stock Reagent | Typical Storage |
|---|---|
| 30–40% Acrylamide/Bis (29:1) | 4°C, dark bottle |
| 1.5 M Tris-HCl pH 8.8 | Room temperature |
| 0.5 M Tris-HCl pH 6.8 | Room temperature |
| 10% SDS | Room temperature |
| 10% APS (fresh weekly) | 4°C |
| TEMED | 4°C, tightly sealed |
🧫 SDS-PAGE Gel Recipe
Protein Separation Range Guide
How to Use the SDS-PAGE Gel Calculator
This free online calculator computes exact reagent volumes for both the resolving and stacking gels used in SDS-PAGE (Sodium Dodecyl Sulphate–Polyacrylamide Gel Electrophoresis). Follow the steps below to generate a precise, ready-to-use recipe for your lab bench.
Step-by-Step Instructions
- Select a resolving gel acrylamide percentage. Use one of the quick preset buttons (8%, 10%, 12%, 15%, 18%) or type a custom value between 6% and 20% into the input field. The preset buttons populate the field automatically and highlight the protein separation range that percentage is best suited for.
- Enter the resolving gel volume. Type the total volume of resolving gel you need in millilitres. For standard mini-gel cassettes (e.g. Bio-Rad Mini-PROTEAN), 10 mL is typical. For large-format gels, use 30–60 mL.
- Enter the stacking gel volume. For mini-gels, 3 mL is standard. The stacking gel is always prepared at 5% acrylamide regardless of the resolving gel percentage chosen.
- Set the number of gels. If you are casting multiple gels at once, enter that number here. All component volumes in the output are automatically multiplied by this factor so you can prepare a single master mix.
- Select your acrylamide stock concentration. Choose 30% or 40% depending on the ready-made acrylamide/bis solution in your lab. Most labs use 30% (29:1 ratio); 40% stock reduces the acrylamide volume needed.
- Select your APS stock concentration. If you prepare fresh 10% APS weekly, select 10%. If your lab uses 5% APS, select that option instead. The calculator adjusts the APS volume so the final amount in the gel is identical either way.
- Click Calculate Gel Recipe. The calculator instantly outputs a complete ingredient table for both the resolving and stacking gels, with volumes rounded to three decimal places (µL precision).
The Scientific Formula Behind the Calculation
The calculator uses the standard Laemmli SDS-PAGE recipe, which distributes the total gel volume among five components in fixed proportions, with acrylamide adjusted for the chosen percentage:
- Acrylamide/Bis volume = (Target % ÷ Stock %) × Total volume — this scales the acrylamide volume proportionally to achieve the desired final percentage.
- 1.5 M Tris-HCl pH 8.8 = Total volume × 0.25 — provides 25% of total volume for the resolving gel buffer, yielding a final Tris concentration of ~375 mM.
- 10% SDS = Total volume × 0.01 — gives a final SDS concentration of 0.1% w/v, which ensures all proteins remain denatured and negatively charged throughout migration.
- APS = Total volume × 0.01 (10% stock) or × 0.02 (5% stock) — delivers the free radical initiator at the correct final concentration regardless of stock dilution.
- TEMED = Total volume × 0.001 — added at 0.1% v/v as the polymerisation catalyst.
- Distilled water = Total volume − (sum of all other components) — makes up the balance so the total volume is exact.
For the stacking gel, the same formula applies but the target acrylamide percentage is fixed at 5%, and the Tris buffer used is 0.5 M Tris-HCl pH 6.8 at 25% of the stacking gel volume — resulting in a final Tris concentration of ~125 mM at the lower pH required for the stacking mechanism.
When to Use This Calculator
Use this tool whenever you are preparing SDS-PAGE gels from scratch in the laboratory. Common scenarios include: running Western blots for protein expression analysis, checking recombinant protein purity after chromatography, comparing molecular weights of unknown bands against a protein ladder, and monitoring protein cleavage or modification. The multi-gel scaling feature is especially useful when running several cassettes simultaneously — a common requirement in labs doing high-throughput expression screening or antibody validation.
Common Mistakes to Avoid
- Using old APS. Ammonium persulfate degrades quickly once dissolved. A 10% APS solution stored at 4°C should be replaced weekly. Old APS produces slow, incomplete, or uneven polymerisation, resulting in a gel that never fully sets or runs with distorted band patterns.
- Adding APS and TEMED too early. Polymerisation begins the moment APS and TEMED contact the acrylamide. Both should be added as the very last step, and the gel should be poured immediately after mixing. Preparing other reagents or setting up the casting stand after adding APS and TEMED will lead to partial gel setting in the vessel.
- Not degassing the water. Dissolved oxygen inhibits free radical polymerisation. For high-quality gels with consistent pore sizes, degas the distilled water (and acrylamide mixture before adding APS/TEMED) under vacuum for 10–15 minutes before use. This is especially important for high-percentage gels (15–18%) where polymerisation kinetics are more sensitive.
- Incorrect stock concentration selection. Using the wrong stock concentration in this calculator will produce incorrect acrylamide volumes, yielding a gel that runs at the wrong effective percentage. Always verify whether your acrylamide/bis bottle is labelled 30% or 40% before entering values.
- Not allowing the resolving gel to polymerise fully before pouring the stacking gel. The resolving gel needs at least 30–45 minutes at room temperature to polymerise completely. Pouring the stacking gel too early traps residual acrylamide from the resolving layer into the stacking interface, blurring the boundary and reducing band sharpness.
Interpreting Your Results
The output tables list volumes in millilitres (mL) to three decimal places, already multiplied by the number of gels you specified. Mix all components in the order listed — water first, then acrylamide, then buffer, then SDS — before finally adding APS and TEMED. The separation range guide below the output highlights which protein size range your chosen resolving gel percentage is best calibrated for, giving you a quick visual confirmation that your acrylamide selection is appropriate for your target protein.
Frequently Asked Questions
What acrylamide percentage should I use for my protein?
The optimal acrylamide percentage depends on your protein's molecular weight. Use 8% for large proteins between 40 and 200 kDa, 10% for medium-range proteins from 20 to 120 kDa, 12% for proteins in the 10–70 kDa range, 15% for small proteins between 5 and 40 kDa, and 18% for very small proteins or peptides from 3 to 20 kDa. When working with a mixture of proteins spanning a wide size range, consider a gradient gel such as 4–20% pre-cast acrylamide, which cannot be calculated with this tool but is widely available commercially.
Why is the stacking gel always 5% acrylamide regardless of my resolving gel percentage?
The stacking gel operates on a different principle from the resolving gel. At 5% acrylamide, the large pore size allows all proteins to migrate freely. Combined with the lower pH of 6.8 and the discontinuous buffer system (Laemmli system), the stacking gel concentrates all proteins into a sharp, thin starting zone before they enter the resolving gel. This stacking effect, driven by the isotachophoretic migration of glycinate ions, dramatically improves band resolution in the resolving gel. Changing the stacking gel percentage would compromise this concentration mechanism.
How do I calculate how much APS to use, and does the APS stock concentration matter?
APS (ammonium persulfate) initiates polymerisation by generating free radicals. The volume used depends on the APS stock concentration you have prepared: for a 10% APS stock, use 1% of the total gel volume (0.01 × volume); for a 5% APS stock, use 2% of the gel volume (0.02 × volume). Both deliver the same final APS amount in the gel. This calculator automatically adjusts the APS volume based on your selected stock concentration. Always prepare fresh 10% APS weekly — old APS leads to slow or incomplete polymerisation, resulting in soft or uneven gels.
Why should APS and TEMED be added last, and how quickly must I pour the gel?
APS provides the free radicals that initiate polymerisation, and TEMED (tetramethylethylenediamine) acts as the catalyst that stabilises those radicals to propagate the chain reaction through the acrylamide monomers. Once both are added together to the gel mixture, polymerisation begins immediately. At room temperature, you typically have 5–10 minutes before the mixture becomes too viscous to pour evenly. Delay will cause uneven polymerisation, bubbles, or a gel that sets partially in the beaker. Pre-chill all solutions to 4°C if you need more working time, especially for high-percentage gels.
What is the difference between 30% and 40% acrylamide stock, and does it affect the recipe?
Acrylamide stock solutions are supplied at either 30% or 40% total acrylamide with a fixed acrylamide-to-bis-acrylamide ratio (most commonly 29:1). Using a 40% stock means you need less volume of it to achieve the same final acrylamide concentration in the gel, so all other component volumes remain proportionally larger — this is reflected automatically in this calculator when you switch between stock concentrations. The final gel composition and resolution are identical regardless of which stock you start from, as long as the acrylamide-to-bis ratio is the same. Most labs use 30% stock for convenience, as it reduces pipetting error at low acrylamide percentages.