Day: July 18, 2020

The immunoassays are widely used techniques in both basic research and diagnostic and clinical applications. The suitability of the antibodies used is critical in obtaining the expected result, and this does not only affect the primary antibody (which will bind to the antigen of interest), but also the secondary antibody that will allow us to detect this binding.

Therefore, it is necessary to pay attention to some basic aspects that will help us select the most appropriate secondary antibodies for each case.

Selecting Secondary Antibodies: Criteria To Consider

1. “Host” of the primary antibody: 

The secondary antibody must be directed against the species of the primary, so it is essential to know this information. If the primary antibody was produced, for example, in mouse, the secondary one must be an anti-mouse obtained in a different species than this one.

2. Experimental procedure or technique in which it will be used:

This information is essential when choosing the labeling / conjugation of our secondary antibody.

• When detection is carried out by enzymatic reaction, as in the case of techniques such as Western Blot or ELISA immunoassay , secondary antibodies must be labeled with an enzyme (Alkaline phosphatase (AP), Peroxidase (HRP) …) or conjugated to biotin for amplification in two steps.
• When detection is carried out by fluorescence, as occurs in Flow Cytometry (FC), Immunofluorescence (IF), Immunohistochemistry (IHC), Immunocytochemistry (ICC) …, the secondary antibodies must be marked with a fluorochrome.

3. Isotype and subclass of the primary antibody:

The secondary antibody will be directed against the isotype of the primary antibody. Although the vast majority of primary antibodies (especially polyclonal antibodies ) are IgGs, this is an aspect to pay attention to, being essential to know the isotype of the primary antibody, and its subclass is recommended if there is one.

As a reminder, we leave you a summary with the different isotypes of immunoglobulins according to the most frequent species:

4. Purification level of secondary antibody

Secondary antibodies can be presented as an IgG fraction, or as affinity purified antibodies. Each of these two formats has its advantages and disadvantages, which should be known in order to make the most appropriate choice in each case.

• IgG fraction: The main advantage of this presentation is that the high affinity of these antibodies will allow for greater signal amplification, facilitating the reading of the results. In return, the specificity is lower, and may lead to nonspecific junctions that will result in high background noise.
• Affinity purified antibodies: The main advantages in this case would be the high specificity (with the consequent low background level), high sensitivity and high reproducibility of immunoassays. All this to the detriment of affinity, and may lead to weaker reactions.

We hope this short guide has been helpful in helping you select the secondary antibodies that best suit your assay.

Research in the field of proteomics can cover aspects as diverse as structure, function, modifications, location or interactions between proteins. In any of these fields, a source is usually required from which to obtain a sufficient quantity of the protein of interest for the study.

Chemical synthesis, valid for small peptides , is not a viable option for complete proteins, due to its size and structural complexity. That is why, in the absence of a native protein source, the expression of recombinant proteins is frequently resorted to .

The process of obtaining heterologous proteins basically consists of cloning the gene encoding the protein of interest in an organism other than the one that originally produces it:

However, the expression of recombinant proteins , like any other biotechnological process, is subject to the particularities of each protein in particular, having to solve in each case some challenges such as:

• Solubility
• Conformation
• performance
• Purity
• Additional difficulties for toxic membrane proteins that degrade easily …

Factors That Impact The Expression Of Recombinant Proteins

Countless factors can directly influence viability and performance when expressing proteins recombinantly. Here we summarize some of the most relevant:

1. Sequence : the same protein can be encoded by different sequences forming different codons that give rise to the same amino acid. The variability in the level of expression between two different codons that code for the same amino acid can be up to 250 times. Therefore, before synthesizing the DNA of interest for the expression of our recombinant protein, it is of utmost importance to carry out a codon optimization process that allows us to select the most appropriate sequence.

2. Vector : each of the vector’s structural units can influence the expression performance of a recombinant protein, from the ribosome binding sequences (RBS) to the sequences that encode the tag. But among all of them, the promoter sequence is of special relevance. Note that although generally the stronger the promoter, the greater the performance of the expression, there are exceptions. For example, in the case of toxic proteins, a weak promoter is expected to improve performance.

3. Expression system : the selection of the expression system is key for the expression of the recombinant protein to be successful. This selection should be based on the following criteria:

• Protein characteristics, such as type (membrane, cytoplasmic …), molecular weight, post-translational modifications …
• Application: structural biology, functional assays, antigens for the production of antibodies, protein-protein interaction studies, therapeutic applications …
• Yield to be obtained
• Cost or budget available

We leave you a summary with the strengths and weaknesses of the most common expression systems:

4. Strain (in case of prokaryotes) or cell line (in eukaryotes): once the expression system has been selected, it is necessary to pay attention to the specific strain / cell line to be used, since these can directly influence aspects such as the formation of disulfide bridges or in the reduction of cellular toxicity.

5. Expression conditions : it is also recommended to carry out a small expression test to optimize the different conditions that can affect performance such as the composition of the medium, the temperature, the concentration of the inductors and the induction time or the inoculation volume between others.

The expression of recombinant proteins is a complex process, which requires setting many variables to optimize performance and obtain soluble and functional proteins. We hope that these brief brushstrokes have helped you to clear some doubts.

You can consult our recombinant protein expression service here , and do not hesitate to contact us to ask us any questions in which we can advise you.