Total Protein Assay as a Step in Peptidomics Analytical Frame Work

George, Rohi; Sweedler, Jonathan V.

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https://hdl.handle.net/2142/99935 Copy

Description

Title

Total Protein Assay as a Step in Peptidomics Analytical Frame Work

Author(s)

• George, Rohi
• Sweedler, Jonathan V.

Contributor(s)

Romanova, Elena V

Issue Date

2018-04

Keyword(s)

• Chemistry
• Peptidomics
• BCA protein assay
• mouse brain

Abstract

Peptidomics is the complete quantitative and qualitative analysis of endogenous peptides in a biological sample. The total protein content is an important analytical task for peptidomics as it allows for the determination of the sample size required for such analysis. One particular method that is used for the quantitation of total protein is the BCA method. The BCA method uses the reaction of two cuprous ions with a bicinchoninic acid (BCA) molecule to produce a purple colored product. In addition, the BCA/copper complex absorbs at a wavelength of 562 nm, and this wavelength is used to detect the absorption of the sample. For this particular project, the Thermo ScientificTM Micro BCATM Protein Assay Kit was used to assay the total protein in frozen, pulverized mouse brain tissue. The kit used diluted protein standards and concentrated reagents along with a period of incubation at 37°C to produce a colorimetric protein assay in a 384-well plate. Using the Epoch microplate spectrophotometer, a wavelength of 562 nm was selected to interact with the samples, and the number of photons absorbed by the samples was detected by the photometer. The data was collected by the Gen5 software, and a standard curve was prepared by plotting the absorbance against concentration. This curve was used to determine the concentration of protein in the unknown mouse brain samples.

Type of Resource

image

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http://hdl.handle.net/2142/99935

Sponsor(s)/Grant Number(s)

• UIUC Neuroproteomics and Neurometabolomics Center on Cell-Cell Signaling
• P30DA018310

Copyright and License Information

• Copyright 2018 Rohi George
• Copyright 2018 Jonathan V. Sweedler

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