Immunoprecipitation for Mass Spectrometry

Example 10

Immunoprecipitations were performed as described above. Briefly, cells were crosslinked with 1% formaldehyde at room temperature for 10 minutes followed by glycine quenching, cell lysis, nuclei isolation and lysis, and brief sonication. Protein complexes were immunoprecipitated overnight using an anti-HA antibody (Cell Signaling 3724), and were washed and eluted as described above. Elutes were used directly for mass spectrometry experiments without further dilution.

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US11293018B2. Manipulation of nuclear architecture (2022-04-05). THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY [US], SYSTEM BIOSCIENCES, INC. [US]. Inventors: Kevin Chun-Kai Wang [US], Stefanie Morgan [US], Fangting Wu [US], Chiao-Chain Huang [US].

Patent 2022

anti-HA antibody

Anti antibody Cell Chromatin immunoprecipitation Dilution Formaldehyde Glycine Isolation Mass spectrometry Nuclei Protein

Top 5 similar protocols

Variable analysis

independent variables

Immunoprecipitation using an anti-HA antibody

dependent variables

Protein complexes identified by mass spectrometry

control variables

Cell crosslinking with 1% formaldehyde
Glycine quenching
Cell lysis
Nuclei isolation and lysis
Brief sonication

controls

Positive control: None mentioned
Negative control: None mentioned

Annotations

Based on most similar protocols

Perform cell lysis and immunoprecipitation at low temperature (4°C) to prevent protein degradation and maintain protein-protein interactions (Protocol 5)

Use appropriate control samples, such as normal IgG or unrelated antibodies, to validate the specificity of the immunoprecipitation (Protocol 4)

Optimize the antibody concentration and incubation time for immunoprecipitation to ensure efficient capture of the target protein (Protocols 3 and 4)

Consider using a non-denaturing lysis buffer, such as the NET gel buffer described in Protocol 3, to maintain the native structure of the protein complexes during immunoprecipitation

Perform crosslinking of cells with formaldehyde prior to immunoprecipitation to stabilize protein-protein interactions and capture transient complexes (Protocols 3 and 5)

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