There are several critical sample preparation steps that affect the downstream MS-analysis and they must be considered before performing the experiment. Please contact the Core Facility to discuss the project prior to sample preparation.
The sample will be concentrated and digested using filter aided sample preparation (FASP). Tryptic digested samples will then be analyzed with nanoLC MS/MS followed by matching against a publicly available protein database as SwissProt using Proteome Discoverer for protein identification.
Optimization of protocol
Detailed protocols and guidance for setting up an IP/pull down experiment are provided by the manufacturer of the beads. In order to optimize experimental conditions, work initially on a small scale. For the final MS-analysis it is important to do a large scale preparation.
We recommend that you optimize your IP by monitoring the efficiency with Western blotting (WB) against your protein. To determine the quality of IP/pull-down and amount of protein in the sample (protein of interest, background proteins and antibody) SDS-PAGE and Coomassie staining are required. Image of WB and Coomassie-stained gel are send together with the sample submission form, when you hand-in the samples to the Proteomics Core Facility.
To reduce the background elute using mild conditions. SDS in elution buffer will give massive background, since non-specifically bound proteins will be eluted together with target proteins. In IP experiments, it is important to cross-link the antibody to the beads. Both background proteins and antibody in the eluate will obstruct identification of the proteins in the complex. PEG-containing detergents as NP40, TritonX100, and Tween should be avoided or used in very low concentrations.
The researcher is responsible for the IP/pull-down optimization and procedure and that it has been performed in a MS-compatible way.