Shotgun identification of protein modifications from protein complexes and lens tissue

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Shotgun identification of protein modifications from protein complexes and lens tissue

The National Academy of Sciences
Publication Date
Jun 11, 2002
  • Biology
  • Chemistry


STANDARD OPERATING PROCEDURE M-106.1 Page 1 of 5 STANDARD OPERATING PROCEDURE Title: Immunohistochemistry (IHC)/Tissue Microarray (TMA) of NCI60 Cell Line Array (CMA) SOP#: M-106 Version #: 1 Author: S. Hewitt Date Approved: Dec. 23, 2009 Date Modified: 1. The purpose of this document is to describe the procedure for immunohistochemistry (IHC), tissue microarray (TMA), and antibody titration. This immunohistochemistry protocol can be used with novel antibodies, but starting titers of antibody must be developed from other information, such as preliminary data from the Well-based "Reverse Phase" Protein Array screen of the NCI60 panel of protein lysates (SOP, M-105). PURPOSE 2. This procedure may be used for general immunohistochemical staining and antibody titration. SCOPE 3. It is the responsibility of person(s) performing this procedure to be familiar with laboratory safety procedures. The interpretation of results must be done by a person trained in the procedure and familiar with such interpretation. RESPONSIBILITIES 4. • Leica Autostainer model XL EQUIPMENT • Dako Autostainer model LV-1 or equivalent Note: The use of automated stainers is not required, however assay reproducibility and throughput is significantly enhanced by their use. • Vegetable Steamer (Black & Decker or equivalent) • Coplin Jars with lids M-106.1 Page 2 of 5 5. • Hitology Reagents: MATERIALS o Xylenes; Mallinckrodt Chemicals, Cat. #8668-16 o Alcohols; Sigma-Aldrich, Cat. #459844 • Antibody Dilutent; Dako, Cat #S0809 • IHC Wash Buffer; Dako, Cat #S3006 o Dilute 1:10 with Deionized Water • End Enzyme Block o 3% peroxidase blocking reagent; Dako, Cat. #S2001 • Protein Block o Protein block serum-free buffer; Dako, Cat. #X0909 • Antigen Retrieval Buffer o Target retrieval high pH; Dako, Cat. #S3307 o Dilute 1:10 wit

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