Brighter Labeling with iFluor Conjugates

Secondary antibodies conjugated with iFluor^® designed for robust and sensitive immunoassays

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We are pleased to announce the release of two new product lines of secondary antibody conjugates. The first is a set of goat anti-mouse IgGs conjugates. The second uses goat anti-rabbit IgGs. Both sets of secondary antibodies have been conjugated with our superior iFluor^® fluaorescent dyes. These secondary antibody conjugates are available in whole IgG format (H+L) and optionally cross-adsorbed (against human, swine and bovine IgG). Our goat anti-mouse IgG conjugates can also be optionally cross-adsorbed against rabbit and horse IgG, while our goat anti-rabbit IgG conjugates can also be optionally cross-adsorbed against mouse and rat IgG. Our iFluor^® secondary antibody conjugates are ideal for use in fluorescent western blotting, flow cytometry, fluorescent microscopy and fluorescent microplate readers. Our secondary antibody conjugates have undergone extensive testing and exhibit strong signal quality with minimal background staining. They have also been shown to be brighter than, and a good replacement for, Alexa Fluor^® secondary antibody conjugates.

 

It starts with high quality antibodies

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Secondary antibodies, as a class, have become an essential component of a vast array of immunoassays. This is because, rather than targeting an antigen or protein directly, secondary antibodies target other (primary) antibodies. What this allows for is very efficient immunolabeling. Consider, instead of labeling each and every primary antibody, only a single secondary antibody (which targets afore mentioned primary antibodies) needs to be labeled. This translates into both saved time and reduced cost, two qualities that are essential in today's increasingly competitive industry.

Given the importance of secondary antibodies, it becomes vital to find the right one. In this regard, when researchers are asked, the majority will name specificity as the most important quality they look for when picking a secondary antibody. From a practical point of view, this makes a lot of sense. High specificity typically translates into better binding of the target substrate and less non-specific binding. What this means from an experimental perspective is that a highly specific secondary antibody results in stronger assay signals and lower noise.

The importance of high specificity extends beyond just secondary antibodies; it is of importance to secondary antibody conjugates as well. Recognizing this point, that high quality conjugates start with high quality antibodies, we have laid out an extensive quality control framework that validates our antibodies throughout the conjugation process. For a glimpse into our Quality Management System (QMS), here are a few steps we take to ensure the best quality antibodies:

1. Our host animals are carefully screened and undergo a validated immunization procedure with experimentally supported incubation times
2. Our total serum undergoes multiple rounds of affinity purification and dialysis to remove unwanted impurities and cell components
3. We offer cross-adsorbing of secondary antibodies to prevent cross-reaction with unwanted primary antibodies
4. Each lot of antibodies is tested through ELISA to ensure good activity before being passed to the conjugation process

Only antibodies that meet strict validation standards and show minimal nonspecific binding will pass our QMS and be used for conjugation. This guarantees that our customers receive the highest quality secondary antibody conjugates.

It continues with superior labeling dyes

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For the labeling dyes, we chose to use our superior iFluor^® product line. This is a series of fluorescent dyes known for their brightness and photostability, as well as their minimal quenching when bound to proteins. Our in-house testing has shown that even when the degree of substitution is high (that is, the dye to protein ratio is high), these dyes will not show significant self-quenching, making them perfect for preparing secondary antibody conjugates.

In addition to minimized self-quenching, another reason we chose the iFluor^® product line is because these fluorescent dyes are fairly robust. Specifically, they can be used in a wide range of assay conditions. For example, iFluor^® 488 shows a stable signal across pH 4-10. This fluorescent stability under varying conditions allows for consistent assay results and flexibility in experimental design.

The last reason we chose the iFluor^® product line is because of its excellent fluorescence quantum yields. Broadly speaking, the fluorescence quantum yield is a measure of the number of photons emitted by a substance in relation to the number of photons it absorbed. When two compounds absorb identical amount of photons then, the one with the higher fluorescence quantum yield will emit more photons and appear brighter. Since our iFluor^® dyes have high fluorescence quantum yields, what this means is that under identical excitation conditions, our secondary antibody conjugates will outperform with better brightness.

Our iFluor^® product series comes in a wide array of excitations and emissions. At the moment, the product line consists of: iFluor^® 350, iFluor^® 405, iFluor^® 488, iFluor^® 514, iFluor^® 532, iFluor^® 555, iFluor^® 594, iFluor^® 610, iFluor^® 633, iFluor^® 647, iFluor^® 660, iFluor^® 680, iFluor^® 700, iFluor^® 750, iFluor^® 750 and iFluor^® 790, ranging the entire spectrum from UV to Near Infrared. Additionally, they are compatible with most standard lasers (eg. 488 nm argon-ion laser), allowing for easy adoption and convenient application.

It ends with conjugation and optimization

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It starts with high quality antibodies. It continues with superior labeling dyes. It ends with conjugation and optimization.

What differentiates our secondary antibody conjugates from others on the market is our comprehensive conjugation and optimization process. Thanks to the collaborative efforts of our chemists and biologists, we are able to tailor our conjugation process to each antibody-dye pair, ensuring optimal results for each one of our conjugates individually. We accomplish this with several key steps.

First, we work to determine the ideal starting conditions for a particular antibody-dye conjugation. This includes developing the best buffer through fine tuning of buffer pH and ionic concentration, two factors which have been shown to influence the conjugation process. Additionally, we experimentally determine the ideal starting concentrations for both the antibody and the labeling dye. Ultimately, our goal for this optimization step is to optimize yield. Research has shown that an ideal starting condition greatly increases yield. An increased yield, in turn, translates into significant cost savings which we then pass to consumers.

Second, we work to determine the ideal conjugation time. On the one hand, there needs to be enough time allotted for conjugation to occur. And not only occur, but occur with high yields which, as previously mentioned, translate into guaranteed cost savings for consumers. On the other hand, conjugation cannot be allowed to occur indefinitely even if it does lead to higher yields. At the very least, it would be impractical. But more importantly, it may lead to decreased antibody activity. That is why we find the sweet spot for conjugation time which maximizes yield while minimizing activity loss.



Third, and perhaps most importantly, we work to determine the ideal dye to antibody ratio, or degree of substitution (DOS). This, too, is a balance. Too little dye labeled onto the antibody leads to poor quality signals and poor brightness. Conversely, too much dye labeled onto the antibody can lead to a variety of problems as well, such as self-quenching, reduced affinity, higher background, poor conjugate solubility, poor conjugate loading and difficulty in usage. That is why our conjugates are carefully prepared to be bright while still maintaining ease of use.

Finally, we work to determine the ideal purification process for our conjugates. Our purification process will remove unlabeled antibody as well as free, unreacted dye. This is a vital step in the process for two reasons. First, unlabeled antibodies will compete with labeled antibodies for binding targets, but they will not produce a signal when bound. This leads to lower quality signals and lower brightness and must be minimized. Second, un-reacted free dye will increase the assay background. While this is detrimental for a variety of reasons, the most important is that it makes assay signals difficult to read and interpret, possibly masking the true positives while generating false ones. For these two reasons, optimal purification is essential to our conjugation process.

As you may have discovered, our optimization is not a single step, but rather, a comprehensive framework which spans the entire conjugation process. Ultimately, our goal is to provide the brightest secondary antibody conjugates at an affordable cost, that researchers may have a powerful tool for use in their studies. Below, you may find a sampling of our current conjugates.