Typical range: 8–18%
Total volume for resolving gel.
Typically 2–4 mL for mini gels.
| 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
Introduction
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.
About the Tool
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), the standard method for separating proteins by molecular weight before Western blotting, purity checks, or expression analysis. Enter your target acrylamide percentage, gel volumes, number of gels, and stock concentrations, and the calculator returns a complete, ready-to-use recipe for your lab bench.
Input Explanation
Each field below controls one part of the final recipe. Understanding what each one does helps you get an accurate, lab-ready result on the first try.
- Resolving Gel Acrylamide %. Use one of the quick preset buttons (8%, 10%, 12%, 15%, 18%) or type a custom value between 6% and 20%. This determines how finely the gel separates proteins by size.
- Resolving Gel Volume (mL). Total volume of resolving gel needed. Standard mini-gel cassettes (e.g. Bio-Rad Mini-PROTEAN) typically use 10 mL; large-format gels use 30–60 mL.
- Stacking Gel Volume (mL). Total volume of stacking gel needed, typically 2–4 mL for mini-gels. The stacking gel is always prepared at 5% acrylamide regardless of the resolving gel percentage chosen.
- Number of Gels. If casting multiple gels at once, enter that count here. All output volumes are automatically multiplied by this factor so you can prepare a single master mix.
- Acrylamide Stock Concentration (%). Choose 30% or 40% to match the ready-made acrylamide/bis solution in your lab. Most labs use 30% (29:1 ratio); 40% stock reduces the acrylamide volume needed.
- APS Stock Concentration (%). Select 10% if you prepare fresh APS weekly, or 5% if that's what your lab stocks. The calculator adjusts the APS volume so the final amount in the gel is identical either way.
Formula Explanation
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, in mL — scales the acrylamide volume proportionally to achieve the desired final percentage.
- 1.5 M Tris-HCl pH 8.8 = Total volume × 0.25, in mL — provides 25% of total volume for the resolving gel buffer, yielding a final Tris concentration of ~375 mM.
- 10% SDS = Total volume × 0.01, in mL — gives a final SDS concentration of 0.1% w/v, keeping proteins denatured and negatively charged throughout migration.
- APS = Total volume × 0.01 (10% stock) or × 0.02 (5% stock), in mL — delivers the free-radical initiator at the correct final concentration regardless of stock dilution.
- TEMED = Total volume × 0.001, in mL — added at 0.1% v/v as the polymerisation catalyst.
- Distilled water = Total volume − (sum of all other components), in mL — 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 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.
Worked Example
Sample Input
10 mL resolving gel at 12% acrylamide, 3 mL stacking gel, 1 gel, 30% acrylamide stock, 10% APS stock.
Step-by-Step Calculation
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
Final Result
4.000 mL acrylamide/bis, 2.500 mL 1.5 M Tris-HCl pH 8.8, 0.100 mL 10% SDS, 0.100 mL 10% APS, 0.010 mL TEMED, and 3.290 mL distilled water — totalling exactly 10 mL of 12% resolving gel.
Interpretation
Mix water, acrylamide, Tris, and SDS first, then add APS and TEMED last, immediately before pouring. This same proportional method scales automatically to any percentage, volume, or gel count you enter above.
Result Interpretation
The output tables list volumes in millilitres to three decimal places, already multiplied by the number of gels 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 a quick visual check that your acrylamide selection matches your target protein.
Practical Applications
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.
Scientific Notes & Limitations
This calculator assumes a standard discontinuous Laemmli buffer system with a fixed 29:1 acrylamide:bis-acrylamide ratio; it is not designed for gradient gels (e.g. 4–20% pre-cast formats), which require a different pouring method and cannot be calculated with a single fixed-percentage formula. Volumes are calculated at room temperature and do not account for reagent-specific density variations, which are negligible for standard SDS-PAGE stocks but can matter for highly viscous custom buffers. As with any lab calculation tool, results should be sanity-checked against your own protocol before use, particularly when working with non-standard Tris pH values or alternative crosslinkers such as bis-acrylamide substitutes.
Practical Tips
- Degas your water. Dissolved oxygen inhibits free-radical polymerisation. Degassing distilled water (and the acrylamide mixture before adding APS/TEMED) under vacuum for 10–15 minutes improves pore consistency, especially for high-percentage gels (15–18%).
- Pre-chill for more working time. If you need extra time to pour, especially for high-percentage gels, pre-chill all solutions to 4°C to slow the polymerisation reaction.
- Let the resolving gel set fully. Allow at least 30–45 minutes at room temperature before pouring the stacking gel, so the interface between layers stays sharp.
- Keep APS fresh. Prepare 10% APS weekly and store at 4°C — old APS causes slow or incomplete polymerisation.
Common Mistakes
- Using old APS. Ammonium persulfate degrades quickly once dissolved. 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 poured immediately after mixing.
- Not degassing the water. Skipping this step is a common source of uneven pore sizes, particularly in high-percentage gels where polymerisation kinetics are more sensitive.
- Incorrect stock concentration selection. Using the wrong stock concentration in this calculator produces 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%.
- Pouring the stacking gel too early. The resolving gel needs at least 30–45 minutes to polymerise fully. Pouring the stacking gel too soon traps residual acrylamide at the interface, blurring the boundary and reducing band sharpness.
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.