Dye Dilutions and Lowry Method for Protein Quantification
Importance of Dye Dilutions
Preparing six different dye dilutions from an unknown stock solution is necessary.
Using the undiluted unknown may lead to values outside the linear range of the standard curve.
Standard curves help determine the linearity according to Beer-Lambert Law.
Standard Curve and Linear Range
Based on the standard curve, one can only use absorbance values of the unknown solution that fall within a specific linear range.
For the discussed example, this range is approximately between and .
Absorbance values outside this range cannot be used, as the linearity is uncertain beyond this scope.
Preparation of Dilutions
Multiple dilutions ensure that at least one or two will fall within the linear range of the standard curve.
Different final volumes for dilutions:
Tubes 7 to 9 are prepared in test tubes.
Tubes 10, 11, and 12 are prepared in volumetric flasks.
Proper technique is required, including ensuring accurate meniscus readings in volumetric flasks.
Calculating Stock Solution Volumes
Before the online quiz, calculate the amount of stock solution needed for each dilution; this information is essential.
Refer to Step 1 of Experiment 2b in the provided manual (page 40).
Pipette one milliliter from each dilution into six separate clean test tubes.
Adding Lowry Reagents
After creating dilutions, do not add Lowry A reagent directly to them.
First, aliquot one milliliter from each dilution into clean test tubes, then add five milliliters of Lowry A.
Role of Lowry Reagents in Protein Quantification
Lowry A contains copper; during a ten-minute incubation post-addition, it forms complexes with proteins.
After ten minutes, Lowry B is added; it contains phospho reagents which facilitate further reactions for protein quantification.
Expect a total incubation time of thirty minutes after the addition of Lowry B to fully complete the reaction.