By using immunoprecipitation in combination with mass spectrometry (IP-MS) it is possible to verify that an antibody interacts specifically with an intended target. Unlike other techniques for assessing antibody specificity, such as identifying the disappearance of a band by western blot staining in samples from a knockout mouse, mass spectrometry directly identifies peptide sequences from the proteins in the sample.


Workflow of antibody validation by IP-MS

The overall workflow for antibody validation by IP-MS is illustrated in the accompanying diagram. After an antibody is chosen for analysis, candidate cell lines are selected as models based on their known ability to express the target protein of interest. Next, an optimized immunoprecipitation from cell lysates is carried out, and the resulting IP eluate samples are then prepared and analyzed by mass spectrometry. Finally, the resulting MS data set is processed using customized bioinformatics analysis software. Briefly, this involves: (1) differentiating true positives from negative controls and background, and (2) calculating fold enrichment to evaluate direct (IP) and indirect (co-IP) products by comparing the abundance and enrichment of the target relative to off-target proteins (see subsequent section).

Workflow for antibody validation by IP-MS

Workflow for antibody validation by IP-MS.


Analysis of IP-MS antibody validation data

IP-MS protein grouping

In a typical validation experiment, IP-MS is carried out with two different antibodies: the antibody of interest, and at least one well-characterized control antibody that is specific to an unrelated target. The abundance (e.g., MS signal intensity) of each protein identified by MS is then plotted with respect to the two IP antibodies. The proteins fall into three distinct groups:

  • Background—proteins that are present in both samples are found in the central diagonal region of the graph. These are background proteins that bind either to antibodies in general or to the resin used in the immunoprecipitation.
  • Negative control—proteins that only bind to the negative control antibody are found along the x-axis (i.e., y-value is essentially zero). These are negative control proteins, which do not interact with the antibody of interest.
  • Positive—proteins that bind only to the antibody of interest are found along the y-axis (i.e., x-value is essentially zero). These are positive proteins, which are specifically immunoprecipitated by the antibody of interest.

 

 

Example of IP-MS data output showing differentiation into three protein groupings: background, negative control, and positive proteins seen only with the antibody being tested.


IP-MS enrichment

Once the positive proteins have been identified, they are plotted to show their fold enrichment by immunoprecipitation relative to the starting cell lysate.

Relative enrichment of proteins after immunoprecipitation and MS analysis

Enrichment of p53 after immunoprecipitation with anti-p53 antibody (Cat. No. MA5-14516) and MS analysis.


Limitations of IP-MS for antibody verification

Not all antibodies will immunoprecipitate proteins from lysates. An antibody might not recognize the native form of its target protein. This does not make the antibody a bad choice for use in other applications. However, it does make it impossible to verify the antibody specificity in IP-MS. Other methods are required to verify these antibodies, including genetic modification (knockout or knockdown), biological activation, and other methods.


Verifying target specificity of Invitrogen antibodies using IP-MS

Invitrogen antibodies that have been verified by IP-MS to bind their target are indicated with a “verified specificity” symbol in search results and on relevant product pages. The graph showing the IP-MS enrichment data is provided on each product page.


*The use or any variation of the word “validation” refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses.