Affinity Purification Mass Spectrometry

Affinity Purification Mass Spectrometry

The overview of Affinity Purification Mass Spectrometry

What is Affinity Purification Mass Spectrometry?

Affinity purification mass spectrometry (AP-MS) is a method used to identify protein-protein interactions in a biological sample. It uses a protein of interest that is "tagged," usually with a molecule that allows for easy purification, and then analyses the proteins that bind to it.

The Process of Affinity Purification Mass Spectrometry

The process begins with a cell lysate being run over a column with an immobilized protein of interest. The proteins that stick to the column are then eluted and analyzed by mass spectrometry to determine their identity.

In-depth understanding of the technique

Stages of Affinity Purification Mass Spectrometry

Stage 1 - Preparation of sample: The cell lysate is prepared, this contains the protein of interest along with other proteins that are present in the cell.

Stage 2 - Immobilization: The protein of interest is immobilized on a column, this allows the proteins that interact with it to bind while others pass through.

Stage 3 - Elution and Analysis: The proteins interacting with the protein of interest are eluted and analyzed by mass spectrometry for their identity.

Real-world Applications of Affinity Purification Mass Spectrometry

AP-MS method is widely used in biological research to study protein function, identify novel protein interactions, and determine the components of protein complexes.

It is also used in drug development to identify proteins that a drug binds to, thereby providing information about a drug's potential efficacy and helping anticipate possible side effects.

In clinical testing, AP-MS can be used to analyze patient samples to identify biomarkers for disease.

Advantages and Challenges of Affinity Purification Mass Spectrometry

Benefits and potential pitfalls of AP-MS

While AP-MS can provide valuable insights into protein interactions, it also comes with challenges. The process can lead to false positives and negatives. However, with proper care and control experiments, these can be mitigated.

Future Perspective of Affinity Purification Mass Spectrometry

As technology improves and our understanding of proteins expands, AP-MS will continue to be a valuable tool in biological research and medicine. We can look forward to its more precise and comprehensive applications.

How to Perform Tandem Affinity Purification Tagging?

Introduction to Tandem Affinity Purification Tagging

What is Tandem Affinity Purification Tagging?

Tandem Affinity Purification (TAP) Tagging is a method used to isolate and purify a specific protein together with its associated proteins (protein complex) from the host organism. This approach uses a bait protein that is genetically fused to a TAP-tag containing two different binding sites, facilitating two-step purification.

Tandem Affinity Purification Tagging: Stage 1

In the first step, the tagged protein, along with its protein complex, is captured from the cell lysate using the first binding site. After thorough washing to remove unbound material, the bound protein complex is eluted from the first binding site.

Tandem Affinity Purification Tagging: Stage 2

In the second purification step, the eluted protein complex binds to the second binding site. This allows for further removal of unspecifically associated proteins. Finally, the bait protein and its interaction partners are eluted. They are then ready for analysis.

Interpreting Results

The bait protein and its interaction partners can be analyzed and identified by mass spectrometry. This analysis allows for the identification of potentially novel interaction partners of the bait protein and could provide new insights into cellular protein functions and interactions.

Practical Considerations

Keep in mind that TAP Tagging technique requires specialized lab equipment, molecular biology skills and can be a time-consuming process. Moreover, it doesn’t cover all possible interactions but provides a good start for exploring protein complexes.

Concluding Tandem Affinity Purification Tagging


Tandem Affinity Purification Tagging is a powerful technique for purifying protein complexes from cells. It provides in-depth knowledge about protein-protein interactions, helping to unravel the complex molecular mechanisms within a cell.

Tandem Affinity Purification Tagging

It is a method for purifying a specific protein along with its associated factors.

TAP-tagging Principle

Fundamental conception of tandem affinity purification.

Vector Construction

The TAP tag sequence is inserted into a cloning vector carrying the target gene.


The genetically engineered vectors are introduced into the desired organism.

Pull down

The TAP-tagged protein and its associates are isolated from cell extracts by affinity purification.

TAP-tagging Components

Individual elements used in tandem affinity purification.

Protein A

Protein A is a highly sticky protein that binds to the antibody-coupled matrix.

TEV protease

TEV protease is responsible for their unique specificity, cleaving the tag from Protein A.

Calmodulin Binding Protein

Calmodulin Binding Protein binds to calmodulin, a protein that facilititates repeateble washing of the sample.

TAP-tagging Applications

How TAP-tagging is used in different areas of biology.

Protein-Protein Interactions

Used to investigate the interaction between proteins in a cellular context.

Protein Complexes

Helps to identify, isolate, and study individual components of protein complexes.

Disease Research

Studying disease-associated proteins and their interactions can lead to therapeutic targets.