Isolation of high quality protein samples from punches of formalin fixed and paraffin embedded tissue blocks.

Author(s): Kroll J, Becker KF, Kuphal S, Hein R, Hofstädter F, Bosserhoff AK

Publication: Histol Histopathol, 2008, Vol. 23, Page 391-5

PubMed ID: 18228195 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of starting material size and degree of crushing on protein extraction from punch biopsies of FFPE tissue specimens.

   Summary of Findings:

   Using the Qproteome FFPE tissue kit, protein was successfully and reproducibly extracted from punch biopsies that were 0.7 mM^3 to 3.5 mM^3. However, the smallest size of punch biopsy showed a reduction in the amounts of high molecular weight proteins MIA2 and E-cadherin. Crushing the punch biopsies was essential for isolation of protein and more extensive crushing lead to the isolation of more protein.

   Biospecimens

   • Tissue - Liver

   Preservative Types

   • Formalin

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	Colorimetric assay
   Protein	1D/2D gels
   Protein	Western blot

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Aliquot size/volume	0.7 mM^3 1.2 mM^3 1.8 mM^3 3.5 mM^3
   Analyte Extraction and Purification	Tissue homogenization	Extensive Medium Minimal
   Western blot Specific	Targeted peptide/protein	MIA2 E-cadherin Beta-actin

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2. Study Purpose

   The purpose of this study was to determine the effects of freeze-thaw cycling of protein in extraction buffer and storage of FFPE specimens for 14 years on the stability of proteins in FFPE tissue.

   Summary of Findings:

   Subjecting the protein in the extraction solution to 3 freeze-thaw cycles significantly reduced the total amount of protein obtained compared with samples subjected to only 1 freeze-thaw cycle. Significantly less protein was extracted from 14 year old FFPE tissue than FFPE tissue that was less than 1 year old (p<0.5).

   Biospecimens

   • Tissue - Skin
   • Tissue - Liver

   Preservative Types

   • Formalin

   Diagnoses:

   • Normal
   • Neoplastic - Melanoma

   Platform:

   Analyte	Technology Platform
   Protein	Colorimetric assay
   Protein	1D/2D gels
   Protein	Western blot

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	Less than 1 y 14 y
   Storage	Freeze/thaw cycling	1 cycle 3 cycles
   Western blot Specific	Targeted peptide/protein	N-cadherin ERK1/2 P-ERK1/2 Beta-actin

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