Pathway Discovery
(Jun Qin, Ph.D., Director)
Services:
- Isolation of protein complexes and MS-based identification of components
- Selection of antibodies and conditions for immuno-affinity isolation of native protein complexes from cell cultures and animal tissues
- Large scale growth of cell cultures used for isolation of protein complexes
- MS-based identification of post-translational modifications of components of isolated complexes
Detailed Description of Technical Services
Pathway Discovery Facility provides an entirely unique proteomic service not offered anywhere else. It offers antibody affinity purification of endogenous protein complexes and MS-based identification and analysis of the associated proteins. This is a one-stop custom service. The Dan L. Duncan Cancer Center investigators are requesting the isolation of protein complexes of their interests. The Pathway Discovery Facility selects a suitable antibody for immunoprecipitation of the desired protein complex and optimizes the cell line, culture conditions, and methods for cell lysis and antibody affinity purification. The complexes are isolated under conditions of cellular activation of appropriate signaling pathways as suggested by the cancer center investigator.
1) Isolation of protein complex
Mass spectrometry (MS) has become a main stream technique in biology research. Mass spectrometry has dramatically improved sensitivity and speed for the identification of proteins, which makes use of mass spectrometric data incorporating database searches to identify proteins. Single protein complex identification is no longer a technical challenge in our facility. The first step of protein complex isolation and identification is pre-screening of cell lines, antibodies and condition. In this step our core carefully invests the cell lines to get the cell lines that express abundant amount of target proteins. In this step we test several different sources of antibodies to check its ability for immune-precipitation. With screened cell lines we are doing large scale cell culture and specific treatment on demand to activate the signaling pathway. Sub-cellular fractionation of proteins and generation of protein extracts are followed by harvesting sufficient amount of cells. We typically use 15 to 25 mg of cytoplasmic fractions, nuclear extracts, membrane bound fractions or chromatin fractions for each large scale immune-precipitation. Isolated protein complexes are analyzed by LC-ESI-LTQ after separating by SDS-PAGE and digestion by specific proteases (trypsin and AspN). The obtained RAW files from LTQ are analyzed by knowledge and experience based data analysis to exclude non-specific binding proteins and identify specific components.
2) Identification of post-transcriptional modification
The identification of posttranslational modification sites of proteins provides a framework to expand the proteins functional regulation, including epigenetic functions. Our core provides PTM analysis service from purified protein complex components by employing our skilful method including highly PTM conserving cell fractionation, efficient protein isolation, sophisticated MS operation and highly trained thoughtful data analysis.
Service Fees
CC Investigator |
BCM and Other Institutions |
|
| Protein complex isolation and detection | ||
| Antibody screening | $200 |
$1000 |
| Immuno-precipitation | $500 |
$2000 |
| Band-by-band LC-ESI-LTQ | $200 |
$350 |
| Specific database analysis | $50 |
$300 |
| PTM analysis | ||
| Immuno-precipitation | $700 |
$3000 |
| Band-by-band LC-ESI-LTQ | $400 |
$1000 |
| Specific database analysis | $100 |
$500 |
| Cell Culture and sample preparation | ||
| Cell culture (1 L) | $300 |
$600 |
| Sub-cellular preparation (10 ml packed cell) | $200 |
$1000 |
Location:
BCM Taub building, Room T316
Director:
Jun Qin, Ph.D.
Associate Professor
Dept. of Biochemistry
Phone: 713-798-1507
E-mail: jqin@bcm.edu
Personnel:
Sung Yun Jung, Ph.D.
Lab manager
Phone: 713-798-1517
E-mail: syjung@bcm.edu