Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections.
We describe and discuss a method of protein extraction for Western blot analysis from formalin-fixed, paraffin-embedded tissue sections. From 5-mm2 50-micron-thick tissue sections, an abundance of proteins could be extracted by incubating the sections in lysis buffer containing 2% sodium dodecyl sulfate (SDS) at 100C for 20 min followed by incubation at 60C for 2 hr. Extracts yielded discernible protein bands ranging from 10 kD to 120 kD as identified by SDS-polyacrylamide gel electrophoresis (PAGE). Western blot analysis successfully detected membrane-bound protein such as E-cadherin, cytosolic protein such as beta-catenin, and nuclear proteins including proliferating cell nuclear antigen (PCNA), mutant-type p53, cyclin D1, cyclin E, and cyclin-dependent kinases (CDKs). With this technique, we could examine cyclin D1 and CDK2 expression in small adenomas compared with cancer tissues and normal mucosa. The simple method of protein extraction described here should make it possible to use large-scale archives of formalin-fixed, paraffin-embedded samples for Western blot analysis, and its application could lead to detailed analysis of protein expression. This new technique should yield valuable information for molecular biology.
Ikeda
K
K
Department of Surgery II, Osaka University Medical School, Osaka, Japan.
Monden
T
T
Kanoh
T
T
Tsujie
M
M
Izawa
H
H
Haba
A
A
Ohnishi
T
T
Sekimoto
M
M
Tomita
N
N
Shiozaki
H
H
Monden
M
M
D000236:Adenoma / Q000737:chemistry; D015153:Blotting, Western / Q000379:methods*; D042846:CDC2-CDC28 Kinases; D015179:Colorectal Neoplasms / Q000737:chemistry; D019938:Cyclin D1 / Q000737:chemistry / Q000302:isolation & purification; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000302:isolation & purification; D005557:Formaldehyde; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry / Q000302:isolation & purification; D010232:Paraffin; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000302:isolation & purification; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D016707:Tissue Fixation
D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D011506:Proteins; D019938:Cyclin D1; D005557:Formaldehyde; D010232:Paraffin; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases