6-1.1 ### Protein Analysis via SDS-PAGE#### Conditions and Measurement:A protein was analyzed for its molecular weight under buffer, pH, and temperature conditions that approximate physiological conditions. Initially, size exclusion measurements indicated a molecular weight of 140,000 g/mol.#### SDS-PAGE Gel Analysis:Upon conducting SDS-PAGE electrophoresis with and without the reducing agent β-mercaptoethanol (BME), distinct patterns emerged:- **Lane A (-BME):** Shows the protein pattern without the reducing agent.- **Lane B (+BME):** Shows the protein pattern with the reducing agent.- **Lane C (STD):** Contains molecular weight standards for comparison. #### Molecular Standards: - Serum Albumin: 67,000 - Ovalbumin: 43,000 - Carbonic Anhydrase: 30,000 - Trypsin Inhibitor: 20,000### Discussion Questions:#### Part A: Native Protein Structure1. **Subunit Linking:** - There are *___*, *___* linked subunits suggested by the *___* pattern in the *absence* of BME.2. **Subunit Stoichiometry:** - The native molecule contains *___* units indicated by the *___* pattern in the *presence* of BME.3. **Bonding in Units:** - The *___* units are paired by at least one *___* bond.Students are tasked with filling in the blanks using terms such as "covalently," "disulfide," "ionic," "hydrogen," "noncovalently," among others, based on observations from the SDS-PAGE results. The goal is to determine the nature of the protein’s subunit composition and bonding characteristics.

SDS (Sodium dodecyl sulfate) breaks non-covalent interactions like hydrogen bonds and ionic bonds within the protein. Parts that were just held together by non-covalent interactions get separated upon the addition of SDS.Beta-mercaptoethanol (BME) breaks the disulfide bonds in the protein.The total MW of our protein is 140,000 g/mol. With just SDS, we get a band near 70,000 MW. Which means two identical subunits of MW near 70,000 were together by non-covalent interactions.Upon addition of BME into the mixture that already contained SDS, we got a band near 30,000 and one near 40,000. Which means that the two identical 70,000 bands were composed of a 30,000 and a 40,000 MW unit that were linked to each other via at least one disulfide bond.Note : There are multiple ways to fill in the blanks correctly using the options given. One correct way is given below.There are two identical, noncovalently linked subunits that are suggested by the pattern of in the absence of BME.The native molecule contains four units that are suggested by the pattern of in the presence of BME.The units are paired by at least one disulfide bond.