This elearning course comprises four interactive modules that were developed to provide a succinct, contextual overview of the four pillars of antibody validation* implemented to test the specificity of Invitrogen antibodies.

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Overview

Researchers need antibodies that bind to the right target and function effectively in their intended applications. Not having the right antibody can have far-reaching effects that include:

  • Lost time and money
  • Compromised experimental results
  • Lack of experimental reproducibility

The Four Pillars of Invitrogen antibody specificity validation* were developed to help minimize poor antibody performance. In addition to our rigorous functional application validation testing, Invitrogen antibodies are assessed using advanced validation methods that go beyond our standard testing. Including these additional steps provide further assurance that our antibodies will bind to their intended targets. The four pillars include these distinct testing methods:

  • Immunoprecipitation and mass spectrometry (IP-MS)
  • Genetic modification
  • Independent antibody verification (IAV)
  • Biological verification

This elearning course was developed to help you better understand the principles behind these specificity testing methods so that you know what questions to ask when choosing antibodies for your research.

Each of the four modules is interactive, and you will have opportunities to test your knowledge along the way. Each module takes approximately 30 minutes to view. The tabs below provide more detailed descriptions and the learning objectives for each module.

The course is free, and you only have to complete a simple form to access all four modules. 

Immunoprecipitation and mass spectrometry (IP-MS)

This is the first in a four-part series describing the four pillars of Invitrogen antibody validation* and methods for verifying the specificity and functionality of antibodies used for research applications.

Upon completion of this module, you will be able to:

  • Explain the rationale for antibody specificity validation using the four pillars
  • Describe the workflow for antibody target validation using IP-MS
  • Explain how genes of interest and cell lines are selected for IP-MS experiments
  • Describe the principles of immunoprecipitation
  • Describe the principles of protein identification and analysis by mass spectrometry

Module run time: ~30 min

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Technical content excerpt

IP-MS is a powerful approach to verify antibody selectivity. This technique is unique among the four pillars because it allows researchers to identify and quantitate all proteins that selectively bind to an antibody, including the intended target protein and any off-target proteins. It also allows for the identification of proteins that interact directly with the antibody’s target protein and may be used to detect post-translational protein modifications, such as phosphorylation events. 

IP-MS approach to verify antibody selectivity

bar graph showing fold enrichment for a number of proteins

Figure 1. Antibody validation using IP-MS. Subsequent to immunoprecipitation using the test antibody, protein mass spectrometry and data analysis to remove common background proteins, enriched proteins must be submitted to the STRING database (http://string-db.org) to identify any known protein interactions. As shown by the fold-enrichment graph, these data indicate that the anti-Stat3 antibody recognizes the target protein, represented by the red line, as expected. Additionally, the IP-MS analysis revealed a number of proteins that are known to interact—at varying degrees—with the target protein.     

Relationship of interacting proteins

string diagram showing interactions between proteins
Figure 2. This STRING diagram is a representation of known STAT3 interacting proteins. 

Genetic modification

This is the second in a four-part series describing the pillars of Invitrogen antibody validation* and methods for verifying the specificity and functionality of antibodies used for research applications.

 

Upon completion of this module, you will be able to:

  • Understand the conceptual basis of antibody target validation using genetic methods
  • Understand the workflow for antibody target validation using genetic methods
  • Discuss knockout assays
  • Discuss knockdown assays

Module run time: ~27 min

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Technical content excerpt

The discussion of genetic modification begins with a brief description of two well-established genetic methods most commonly used to alter protein levels for antibody specificity testing. 

First the module provides an overview of the genome editing process known as the clustered regularly interspaced short palindromic repeats (CRISPR)–associated protein 9 (Cas9), a bacterial adaptive immune system that has been altered for genome editing. This technology has been adapted to create knockout cell models that facilitate antibody specificity validation.

After a brief description of CRISPR, RNA interference (RNAi) will be discussed. RNAi takes advantage of a cell’s natural machinery to effectively knock down expression of a gene of interest. It is a widely used method to validate antibody specificity. For both types of analyses, CRISPR-Cas9 and RNAi, the loss of expression of an antibody’s target protein should correlate with the level of antibody binding. Persistent antibody binding in the absence of target protein expression can be indicative of a lack of specificity.

Antibody validation* using CRISPR-Cas9 technology

Figure 1. CRISPR-Cas9 knockout for production of cell models. This system utilizes a noncoding single guide RNA to “guide” the CRISPR-associated Cas9 endonuclease to its intended target site within the genome where it cleaves the DNA resulting in disruption of downstream target protein expression. A target-specific antibody may then be tested to verify specificity against the known target in control cell lines compared to the knockout cell line.

Antibody validation* using RNA interference (RNAi)

Figure 2. Knockdown of target mRNA for production of cell models. In mammalian cells, short pieces of double-stranded RNA initiate the degradation or knockdown of a specific, targeted cellular mRNA. Different approaches for knocking down protein using RNAi involve transfecting target cells with pools of short-interfering RNA (siRNA), which are synthetic small RNAs or by transfecting cells with short hairpin RNA (shRNA) vectors. shRNA is processed within the cell, and the in vivo–generated siRNA can then target its specific mRNA molecule for degradation. Nontargeting controls can be used to test for antibody specificity of the knockdown in cell lines.

Independent antibody verification (IAV)

This is the third in a four-part series describing the pillars of Invitrogen antibody validation* and methods for verifying the specificity and functionality of antibodies used for research applications.

 

Upon completion of this module, you will be able to:

  • Evaluate the rationale for antibody validation using the four pillars
  • Describe the workflow for antibody target validation using independent antibody verification
  • Explain the importance of using antibodies with distinct epitopes in independent antibody verification
  • Identify suitable applications and assays for independent antibody verification

Module run time: ~17 min

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Technical content excerpt

Utilizing two independent antibodies for the same protein target can be a useful tool when testing antibody specificity.

In the ideal scenario, two antibodies are used that target non-overlapping epitopes of an antigen. Obtaining comparable results from antibodies that recognize independent regions of the same target protein, allows for increased confidence that the antibodies specifically and reliably detect their intended target. 

Independent antibody testing

Figure 1. Assessing antibody specificity using independent antibody verification (IAV). Common applications where independent antibody validation can be useful tool in assessing specificity include multi-lysate western blots, IHC arrays, immunofluorescence of multiple cell lines, immunoprecipitation, and flow cytometry.

Biological verification

This is the last in a four-part series that describes four individual pillars of Invitrogen antibody validation* and methods for verifying the specificity and functionality of antibodies used for research applications.

 

Upon completion of this module, you will be able to:

  • Define the antibody reproducibility crisis in science
  • Explain the contribution of antibody validation to resolving the reproducibility crisis
  • List the four pillars for validation of antibody specificity
  • Discern the conceptual basis of antibody target validation using biological verification methods
  • Understand the workflow for antibody target validation by biological verification
  • Describe the use of cell treatment assays for antibody validation
  • Discuss the use of relative expression experiments for antibody validation
  • Explain the use of neutralization assays for antibody validation
  • Discuss the use of peptide arrays for antibody validation
  • Discuss orthogonal methods for antibody validation

Module run time: ~35 min

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Technical content excerpt

After an overview of the Four Pillars of Invitrogen Antibody Validation, we will describe in detail, the fourth pillar of validation, which emphasizes Biological verification testing using these methods: 1) evaluating the ability of antibodies to measure changes in downstream effects following cell treatment; 2) using naturally occurring relative protein expression to confirm specificity; 3) neutralization by blocking protein activity with antibody binding; 4) evaluation of peptide arrays to test specificity against known protein modifications and 5) correlating data obtained using orthogonal validation approaches, which involves comparative studies performed using both antibody-dependent and independent methods.

The four pillars of Invitrogen antibody validation*

Figure 1. The Four Pillars of Invitrogen antibody specificity validation were developed to help minimize poor antibody performance.

Methods for antibody target validation* using biological verification testing

Figure 2. Summary of the fourth pillar of antibody validation.* The biological verification testing methods are divided into five distinct categories that are represented by the blue boxes. 

*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, or products were validated for clinical or diagnostic use.