A well-based reverse-phase protein array applicable to extracts from formalin-fixed paraffin-embedded tissue.
Author(s): Chung JY, Lee SJ, Kris Y, Braunschweig T, Traicoff JL, Hewitt SM
Publication: Proteomics Clin Appl, 2008, Vol. 2, Page 1539-47
PubMed ID: 21136801 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine the optimal method of protein extraction from FFPE tissue for the recovery of full-length immunoreactive proteins measureable by WBRPPA. FFPE specimens were stored for more than 7 y at room temperature prior to this study.
Summary of Findings:
The optimum methods for protein extraction included incubation in AR lysis buffer with pH 9.9, as opposed to the same buffer at a pH of 6.0, RIPA, or T-PER buffers. Inclusion of 1% SDS gave higher protein yields compared to the inclusion of 2% SDS, several other detergents, or a combination of SDS and triton X-100 to the lysis buffer. The addition of 10% glycerol to the extraction buffer was also beneficial, but deparaffinization made no difference in protein yield. Incubation in lysis buffer at a higher temperature (65 degrees C) as opposed to 4, 25, or 37 degrees C, resulted in a broader spectrum of proteins recovered. AR pretreatment was superior to no AR, and a pressure cooker temperature of 115 degrees C was superior to 100 or 124 degrees C in terms of protein recovery. While longer AR times resulted in increased protein yields, protein quality declined when AR was for more than 30 min in a pressure cooker, as determined by Western blotting using PSA, phospho-protein kinase B (pAKT), Beta-tubulin, alpha-methylacyl-CoA (AMACR), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies. The optimum AR time for recovery of intact protein was determined to be 15 min. The authors state that with these extraction parameters, 90% of the protein extracted from an equal volume of fresh tissue was able to be recovered from FFPE tissue. WBRPPA data generated from protein extracted from LCM FFPE tissue showed high correlation with Western blot data and showed greater sensitivity. Storage of vacuum-sealed plates at room temperature for up to 56 d after protein coating resulted in minimal changes to WBRPPA profiles for PSA.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform Protein Colorimetric assay Protein 1D/2D gels Protein Western blot Protein Reverse phase protein microarray Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method RIPA lysis buffer
T-PER lysis buffer
AR buffer pH 6.0
AR buffer pH 9.9
Analyte Extraction and Purification Antigen retrieval Pressure cooker 5 min
Pressure cooker 10 min
Pressure cooker 15 min
Pressure cooker 30 min
Pressure cooker 60 min
None
Analyte Extraction and Purification Incubation duration/condition 3 h at 4 degrees C
3 h at 25 degrees C
3 h at 37 degrees C
3 h at 65 degrees C
Analyte Extraction and Purification Temperature of heat-induced retrieval 100 degrees C
115 degrees C
124 degrees C
Analyte Extraction and Purification Protein solubilization 1% SDS
2% SDS
NP-40
Triton X-100
CHAPS
CYMAL-7
Octyl-D glucopyranoside detergent
N-dentyl-Beta-D maloside detergent
Combinational detergents with SDS and triton X-100
Addition of 10% glycerol
No glycerol
Analyte Extraction and Purification Deparaffinization Yes
None
Storage Time at room temperature 1 d
3 d
7 d
14 d
28 d
56 d
Reverse phase protein microarray Specific Technology platform Western blot
Western blot Specific Targeted peptide/protein PSA
pAKT
Beta-tubulin
AMACR
GAPDH