You are purifying a protein for the first me and decide to analyze it on SDS PAGE. You run two lanes on  the gel, one with DTT and one without DTT (DTT is a subs tute for Beta-mercaptoethanol that is less  s nky) The lane with DTT yields 2 bands one at 30kD and one at 20kD while the one without yields a  single band at approximately 50 kD.    A. What does this result suggest about the overall protein structure? B. Sketch what may be occurring in this protein natively.

Suppose that you are tasked to determine the protein concentration of an unknown protein solution via Bradford assay. You prepared six solutions of bovine serum albumin (BSA) with different concentrations. The initial concentration of the BSA stock solution is 7.50 mg/mL. Approximately 200 µL of Bradford Reagent was added to each of these solutions and the absorbance at 595 nm was taken after 5 minutes. See the table below for data on the standard solutions. Standard # A595 BSA conc (mg/mL) 0.000 0.158 2 1.125 0.291 2.250 0.372 4 3.375 0.503 5 4.500 6 5.625 0.675 Determine the protein concentration, in mg/mL, of the unknown solution if its absorbance at 595 nm was 0.248. Note: Final answer format must be x.xx (two decimal places). Round off only in the final answer. Do not round off in the middle of calculation.

What is the function of sodium dodecyl sulfate (SDS) in SDS-PAGE? stabilizes the gel matrix, improving resolution during electrophoresis SDS solubilizes proteins to give them uniformly negative charges, so the separation is based purely on size. SDS raises the pH of the gel, separating multiunit proteins into individual subunits. SDS solubilizes proteins to give them uniformly positive charges, so separation is based purely on pH.

(a) 1 Normalized fluorescence 0.8 0.6 0.4 0.2 0 50 55 OM 0.100 M 0.200 M 0.300 M 0.500 M 1.00 M 2.00 M 60 113588 65 Temp. (°C) 70 75 80 Where is fully folded protein? • Where is fully unfolded protein? • Where is partially folded protein? • To what does SYPRO orange bind? • Why does fluorescence increase as a function of temperature? ● Define a melting temperature for a protein. • Demonstrate how an estimated melting temperature of the protein in zero molar ligand can be determined. • What is the effect of increasing the molar concentration on melting temperature for this protein? • Why is melting temperature a useful measurement to make for a protein especially if you are interested in protein aggregation?