BIOL3612 Lab 2A: Electrophoresis of Isolated DNA & Protein Lowry Assay

Lab 2A will involve both both protein spectrophotometry and DNA gel electrophoresis. The quiz will be based on the protein spectrophotometry portion.

Lowry Protein Assay

Involves a reaction of protein with cupric sulfate in alkaline solution, resulting in the formation of tetradentate copper-protein complexes that is measured at 650 nm on the spectrophotometer and normalized at 405 nm

Why are we using the Lowry Protein Assay over other forms of assays?

1. The proteins are treated with copper in alkaline solution which allows the reaction to proceed quickly (10 min!)

2. The proteins (through their aromatic side chains on phenylalanine and tyrosine) reduce the Folin reagent to produce a blue-colored product that can be detected at 650 nm (this is also a fast reaction that proceeds for 30 mins)

What is the minimum volume of sample and reagent used to set up an assay for pedestal measurements?

10 uL of sample into 40 uL of reagent for a total volume of 50 uL

Standard

• A solution of a known concentration that is used to calibrate or certify that an instrument is working within acceptable, pre-defined guidelines 

  • Also refers to protein solutions of a known concentration used to define a standard curve 

Standard Curves for Lowry Protein Assay

Absorbance values are compared to the known concentrations established on the standard curve to establish the mathematical relationship between light absorbance (at 650 nm) and protein concentration. The A650 values for unknown protein samples can then be measured and the corresponding protein concentrations

• Works by unlocking the predictable relationship between absorbance and concentration (linear line to find the y=mx+b equation and plug in to find the x value)

Control

 A solution that produces an expected result within a specific range if the “system” is working as expected

• Are valid to use as long as the instrument is calibrated and the control product itself is within the expected concentration range stated in the manufacturer’s specifications 

System

Includes the instrument, protocols being used, techniques employed by the user and the solution utilized as the control 

What do negative values on the spectra indicate and how should we fix it?

• Negative values associated with some spectra indicate that either the pedestals were very dirty when the blank measurement was made or that a sample was used to make a blank or reblank measurement 

• Suggestion: Clean pedestal and measure new blank

What does a jagged appearance throughout a spectrum indicate and how do we fix it?

• A jagged appearance throughout a spectrum may indicate a broken column

• Suggestion: Clean pedestal and recondition the pedestal surfaces and measure new blank. Increase sample volume to ensure proper column formation

What does a reported concentration value of NaN or Out of Range indicate and how do we fix it?

• A reported concentration value of NaN or Out of Range indicates that the sample concentration is outside of the range of the measured standard curve 

• Suggestion: Always include standards that will cover the expected range of the samples prior to sample measurement