> See also: > - Reference # SDS-PAGE > [!summary] **Reagents/Components** > - **Sodium dodecyl sulfate (SDS):** Denatures the protein and gives it a negative charge > - **Polyacrylamide Gel Electrophoresis (PAGE):** Forms long polymers and the gel structure > - **Bis-Acrylamide (BIS):** Cross-links acrylamide polymers > - **APS:** Donates a radcical to initiate gel polymerization > - **TEMED:** The catalyst that reacts with APS radicals to polymerize acrylamide > - **$\beta$-Mercaptoethanol (BME):** Breaks disulfide bonds > - **Bromophenol Blue:** Tracking dye to help loading and running > - **Glycerol:** Weighs down samples in the gel wells > - **Loading Controls:** Ensures each lane had the same amount of sample loaded into it ![[Pasted image 20230310232132.png|300]] - The charge-to-mass ratio are nearly identical for different proteins when SDS is used - SDS coating contributes such a large negative charge to the protein that any minimal positive or negative charges that were present are ### Gel Matrix (Polyacrylamide) The *gel matrix* used for SDS-PAGE is **polyacrylamide**. As with most polymers, acrylamide will not polymerize on its own and requires a [[Radical Reactions|radical reaction]] for the polymer to form. - Two common *catalysts* are **APS** which decomposes to form free radicals and **TEMED** (a skin irritant) which is used to stabilize the free radicals. It's important for the gel matrix to contain **pores** for the molecules being testing to travel through. The **porosity level** can be adjusted to better suit the size of the proteins being studied. - **Bis-acrylamide** is a cross linker molecule that can link separate segments of a polyacrylamide complex. --- A discontinuous system can be created using multiple gel types. Most often, a stacking gel - **Stacking Gel** - Due to the low percentage of acrylamide (highly porous), the proteins will quickly flow through and 'stack' up when they hit the more concentrated resolving gel and begin traveling through it at the same time. - This prevents the *position* of the molecules within the *initial well* from impacting where it ends up in the final gel. - **Resolving Gel** - A higher pH in the resolving gel causes the glycerol in the sample loading buffer to become negatively charged in this region. - This causes a voltage gradient to form across the resolving gel ![[Pasted image 20230301160938.png|300]] When a stacking gel ### Sample Preparation & Loading Buffers The indicator dye (bromoethenol blue) does not interact with the protein sample. - It is expected to run through the gel faster than the protein sample and be a good indicator of when to stop running the current The most commonly used buffer for SDS-PAGE is known as the **sample loading buffer** - Also known as the *Luemmli Buffer* which was developed in 1970 and is the 2nd most cited paper. SDS is only effective at destroying non-covalent bonds. **$\beta$-mercaptoethanol (B-ME)** can be used alongside SDS to break covalent bonds, specifically the disulfide bonds formed during protein folding. Loading controls can be used when running a gel to help ensure that any observed variations in target protein abundance are due to relevant biological variation rather than inconsistencies (incorrect dispension, etc.) in the amount of protein loaded into the gel. ### Lane Markers In order to interpret the size of the proteins on the gel after electrophoresis, markers will be added to a lane within the gel. - These molecular weight markers consist of a precise mixture of stained proteins of a known molecular weight (acting as a standard) - The rate at which samples flow through a gel can depend on a variety of factors (gel concentration, pH, voltage) so a standard marker is necessary to get useful information out of the experiment ![[Pasted image 20230302124957.png|200]] QBM Lab: 54 kDa (GST-EGFP Fusion) 26-27 kDa (GST & EGFP Separate) ### Post-Experiment Protein Visualization TCE (2,2,2-Trichloroethanol) is a relatively new reagent that binds to the tryptophan amino acids present within proteins. - When UV light is shined onto a gel where TCE was added, the proteins will fluoresce - Unlike other protein staining methods (methanol, coomassie), this does not permanently fix the protein to the gel, allowing it to still be used to perform a [[Antibody-Based Lab Techniques|western blot]] transfer