Isolation and solubilization of proteins after TRIzol extraction of RNA and DNA from patient material following prolonged storage.

Author(s): Hummon AB, Lim SR, Difilippantonio MJ, Ried T

Publication: Biotechniques, 2007, Vol. 42, Page 467-70, 472

PubMed ID: 17489233 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to assess protein recovery efficiency in samples stored for 3 years at -20 degrees C as phenol-ethanol supernatants following DNA and RNA TRIZOL-mediated isolation. Three protein solubilization methods were investigated, as well as numerous combinations and concentrations of solvents (urea and SDS).

   Summary of Findings:

   A greater quantity of high molecular weight proteins was obtained after sample dialysis compared to protein yields after precipitation. The 100 uL urea with 100 uL SDS solvent was superior to the others examined for protein solubilization.

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • RNAlater

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Protein	1D/2D gels

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Protein solubilization	Dialysis Precipitation Urea SDS Urea and SDS (1:1)

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2. Study Purpose

   The purpose of this study was to assess protein integrity in samples stored for 3 days at -20 degrees C as phenol-ethanol supernatants after DNA and RNA TRIZOL isolation via Western blot analysis for gamma-tubulin and c-Myc.

   Summary of Findings:

   Protein was successfully recovered from the three specimens analyzed, and ranged in size from 5 to 200 kDa. As determined by Western blot analysis for gamma-tubulin, protein levels were comparable to those observed in the HCT 116 cell line. Western analysis of c-Myc revealed both phosphorlyated and unphosphorlyated forms were present in archived specimens.

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • RNAlater

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Protein	Western blot

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	0 days 3 days
   Analyte Extraction and Purification	Protein solubilization	Dialysis Urea and SDS (1:1)

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