# SDS-PAGE Protocol Overview > Published on Vicena | [View on web](https://vicena.ai/wiki/sds-page-protocol-overview--MS5yg) --- ## User What is the complete protocol for SDS-PAGE including how to prepare resolving and stacking gels, sample preparation with loading buffer, running conditions, and Coomassie staining? --- ## Vicena This protocol describes the standard discontinuous SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) method, often referred to as the Laemmli method, for separating proteins by molecular weight. ### 1. Gel Preparation (Resolving and Stacking) The system uses two gels with different pH levels and acrylamide concentrations to "stack" proteins into a tight band before separation. #### **A. Resolving Gel (Bottom Gel, pH 8.8)** Select the acrylamide percentage based on the target protein size (e.g., 10% for 15–100 kDa, 12% for 10–70 kDa). * **Standard 10% Recipe (for 10 mL):** * $\ce{dH2O}$: 4.0 mL * 30% Acrylamide/Bis-acrylamide mix: 3.3 mL * 1.5 M Tris-HCl (pH 8.8): 2.5 mL * 10% SDS: 0.1 mL * 10% Ammonium Persulfate (APS): 0.1 mL (added last) * TEMED: 0.01 mL (added last) * **Procedure:** Mix ingredients, add APS/TEMED, and pour into the glass cassette, leaving ~2 cm at the top. Overlay with isopropanol or water to ensure a flat surface and exclude oxygen. Polymerize for 45–60 minutes. #### **B. Stacking Gel (Top Gel, pH 6.8)** * **Standard 4% Recipe (for 5 mL):** * $\ce{dH2O}$: 3.0 mL * 30% Acrylamide/Bis-acrylamide mix: 0.67 mL * 0.5 M Tris-HCl (pH 6.8): 1.25 mL * 10% SDS: 0.05 mL * 10% APS: 0.05 mL * TEMED: 0.005 mL * **Procedure:** Remove the overlay from the resolving gel and rinse with $\ce{dH2O}$. Pour the stacking mix and immediately insert the comb. Polymerize for 30 minutes. --- ### 2. Sample Preparation Proteins must be denatured and coated with a uniform negative charge. * **4X Laemmli Loading Buffer:** 200 mM Tris-HCl (pH 6.8), 8% SDS, 40% Glycerol, 0.4% Bromophenol Blue, and 10% $\beta$-mercaptoethanol (BME) or DTT as a reducing agent. * **Protocol:** 1. Mix 3 parts protein sample with 1 part 4X buffer. 2. **Heat:** Incubate at 95°C for 5 minutes (or 70°C for 10 minutes) to ensure complete denaturation. 3. **Centrifuge:** Spin briefly to collect condensation before loading. --- ### 3. Running Conditions 1. **Buffer:** Fill the tank with 1X Running Buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS). 2. **Loading:** Load 10–30 $\mu$g of protein per lane. Include a molecular weight marker in the first or last lane. 3. **Electrophoresis:** * **Initial:** Run at 80–100 V while samples are in the stacking gel. * **Separation:** Increase to 120–150 V once samples enter the resolving gel. * **Duration:** Stop when the blue dye front reaches the bottom of the gel (approx. 45–90 minutes). --- ### 4. Coomassie Staining This visualizes protein bands by binding the Coomassie Brilliant Blue R-250 dye to basic amino acids. 1. **Fix/Stain:** Submerge the gel in Coomassie Stain (0.1% Coomassie R-250, 50% Methanol, 10% Glacial Acetic Acid) for 1 hour to overnight with gentle shaking. 2. **Destain:** Transfer the gel to Destaining Solution (40% Methanol, 10% Glacial Acetic Acid). Change the solution 2–3 times until the background is clear and protein bands are distinct. 3. **Storage:** Store the gel in 5% Acetic Acid or $\ce{dH2O}$ for imaging. **References:** * [SDS-PAGE Guide - Abcam](https://www.abcam.com/en-us/knowledge-center/western-blot/sds-page-guide) * [Performing Protein Electrophoresis - Bio-Rad](https://www.bio-rad.com/en-us/applications-technologies/performing-protein-electrophoresis?ID=LUSPFBNEL) * [Coomassie Staining Protocols - Thermo Fisher](https://www.thermofisher.com/us/en/home/references/protocols/proteins-expression-isolation-and-analysis/staining-protein-gels/coomassie-staining.html) ---