SDS-PAGE Gel Preparation Calculator (Resolving Gel & Stacking Gel)
Formula and Principles
This tool is based on the Laemmli discontinuous SDS-PAGE system (Laemmli UK, Nature 1970;227:680–685, PMID 5432063), the most widely used system for protein electrophoresis and Western blotting. Each gel consists of two layers:
- Stacking gel: 5% acrylamide, pH 6.8. Compresses proteins loaded in each lane into a single, sharp horizontal band before they enter the resolving gel.
- Resolving gel: User-selected acrylamide percentage, pH 8.8. Separates proteins according to the hydrodynamic radius of their SDS–protein complexes (i.e., by molecular weight).
Core Proportions
Each component is prepared as a fixed fraction of the total volume; final concentrations are independent of total volume.
Resolving gel (total volume V_r mL, target concentration T%):
30% Acrylamide/Bis (29:1) V_r × T / 30 mL → final concentration T%
1.5 M Tris-HCl pH 8.8 V_r × 1/4 mL → final concentration 0.375 M
10% SDS V_r × 1/100 mL → final concentration 0.1%
10% APS V_r × 1/100 mL → final concentration 0.1%
TEMED V_r × 1/2500 mL → final concentration 0.04%
ddH₂O bring to V_r
Stacking gel (total volume V_s mL, fixed at 5%):
30% Acrylamide/Bis (29:1) V_s × 1/6 mL → final concentration 5%
1.0 M Tris-HCl pH 6.8 V_s × 1/8 mL → final concentration 0.125 M
10% SDS V_s × 1/100 mL → final concentration 0.1%
10% APS V_s × 1/100 mL → final concentration 0.1%
TEMED V_s × 1/1000 mL → final concentration 0.1%
ddH₂O bring to V_s
Spot-check: A 10% resolving gel, 10 mL total → 30% acrylamide 3.333 mL, 1.5 M Tris 2.500 mL, 10% SDS 0.100 mL, 10% APS 0.100 mL, TEMED 4.0 µL, ddH₂O 3.963 mL. These match the values in standard protocol tables (which typically lump TEMED into the water volume and round water to 4.0 mL). This tool uses exactly the equations above — cross-check against any bench protocol you have on hand.
Resolving Gel Percentage Guide
| Target protein MW | Recommended % |
|---|---|
| > 100 kDa (EGFR 170 kDa, cytoskeletal proteins) | 6–8% |
| 40–100 kDa (p53 53 kDa, HSP70) | 8–10% |
| 20–80 kDa (β-actin 42 kDa, GAPDH 37 kDa) | 10–12% |
| 10–40 kDa (cytokines, histones) | 12–15% |
Scope and Limitations
- Supports uniform-concentration resolving gels only; not applicable to gradient gels (e.g., 4–20%). Gradient gels require two solutions at different concentrations mixed in real time during casting with a gradient mixer — the proportions are entirely different.
- Stock concentrations are fixed at 30% Acrylamide/Bis (29:1), 1.5 M Tris pH 8.8, 1.0 M Tris pH 6.8, 10% SDS, and 10% APS. If your lab uses different stock concentrations, scale the volume proportionally; final concentrations remain unchanged.
- Acrylamide percentage is accepted only in the range 6–18%: below 6% the gel is too fragile to handle intact; above 18% it is difficult to cast uniformly. Outside this range the tool refuses to return a result rather than giving an impractical number.
- The stacking gel is fixed at 5%, the standard Laemmli setting. If you need a 3–4% stacking gel (e.g., for very high-molecular-weight complexes), adjust the proportions manually.
Common Mistakes
- Using degraded APS stock. A 10% APS solution stored at room temperature for several days loses free-radical initiating activity, producing a soft or completely unpolymerized gel — with no error message of any kind. Prepare fresh immediately before use, or aliquot into 100–200 µL tubes and store at −20 °C; use each aliquot once.
- Leaving the resolving gel surface exposed to air. Acrylamide polymerization is a free-radical chain reaction; oxygen inhibits it, leaving a soft layer at the surface that blurs the stacking/resolving gel interface. Immediately after casting the resolving gel, overlay ~200 µL of water-saturated isopropanol; pour it off once polymerization is complete.
- Entering total volume instead of per-gel volume. This tool multiplies the per-gel volume by the number of gels to obtain the master-mix total. If you enter the total volume directly, the result will be multiplied by the number of gels a second time.
Safety Note
Unpolymerized acrylamide monomer is a neurotoxin and is classified by IARC as Group 2A (probably carcinogenic to humans) (IARC Monographs Vol. 60). Always wear gloves and work in a ventilated area when handling liquid acrylamide. Polymerized gels are substantially less toxic, but dispose of gel waste according to your institutional guidelines.
Related Tools
For concentration conversions when preparing stock solutions, see Molarity Calculator and Percent Concentration Converter. To estimate band positions by molecular weight, see Gel Band Size Calculator.
FAQ
Why does the resolving gel use pH 8.8 and the stacking gel pH 6.8?
This is the core mechanism of the Laemmli discontinuous buffer system. In the pH 6.8 stacking gel, glycine exists predominantly in its non-ionic form and migrates slowly, creating a low-conductivity zone between the protein bands and the running front that compresses proteins from all lanes into a single sharp horizontal layer. Upon entering the pH 8.8 resolving gel, glycine becomes fully ionized and migrates faster than the SDS–protein complexes; the stacking effect ends and proteins begin to separate by molecular weight.
How do I choose the right acrylamide percentage?
A rough guide: 6–8% for proteins >100 kDa (EGFR 170 kDa, myosin); 8–10% for 40–100 kDa; 10–12% is the most common range, covering β-actin (42 kDa), GAPDH (37 kDa), and p53 (53 kDa) — typical loading controls and targets; 12–15% for small proteins at 10–40 kDa. When in doubt, start with 10%.
Why must APS be freshly prepared or kept frozen?
Ammonium persulfate solution loses its free-radical initiating capacity quickly; a solution stored at room temperature for several days may fail to initiate polymerization, producing a soft or completely unset gel. The problem is that **there is no error signal** — the gel appears to be set until you run it and find streaky or unresolved bands. Prepare a fresh 10% solution immediately before use, or aliquot a fresh batch into 100–200 µL tubes and store at −20 °C; use each tube once and discard to avoid repeated freeze-thaw cycles.
Why does the stacking gel use more TEMED than the resolving gel?
The stacking gel has a small volume and narrow sample wells that need to polymerize quickly to hold their shape. The resolving gel is larger, and slower polymerization actually helps — it allows bubbles to escape and produces a more uniform gel. The final TEMED concentrations are 0.1% (stacking) and 0.04% (resolving), a 2.5-fold difference.
Can I use this tool for gradient gels (e.g., 4–20%)?
No. Gradient gels require two separate solutions at different acrylamide concentrations, combined in real time during casting using a gradient mixer. The volume and composition relationships are entirely different from uniform-concentration gels, and this tool does not handle that case.
Why does the tool refuse to calculate when I enter 20%?
The tool accepts only 6–18%: below 6% the gel is too fragile to peel cleanly from the glass plates; above 18% it is very difficult to cast evenly. Outside that range the tool refuses outright rather than returning a number you cannot practically work with — an impractical result would be worse than no result.
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