Covalent Coupling Troubleshooting & FAQ
Covalent Coupling Troubleshooting & FAQ
My antibody is already purified. Do I really need to purify it again?
Many antibodies are purified using protein A or Protein G affinity columns. During this purification process, tris is often used to elute the antibody off the columns and may be present in varying amounts in the final antibody solution. We strongly encourage purification of all antibodies to remove any possible unknown primary amines which interfere with covalent conjugation.
Do you recommend using MES Buffer for activation?
MES buffer is often used in activation protocols for latex and fluorescent beads to adjust the reaction to pH 5. NanoComposix BioReady™ materials are provided in a mildly buffered aqueous solution that adjusts to pH 5 upon addition of EDC and sulfo-NHS. It is not necessary to activate BioReady™ materials in MES buffer.
Can I just add an excess of antibody rather than performing an optimization?
In many cases, too high or too low of an antibody addition can lead to non-specific binding and a decrease in sensitivity. This is why it is important to empirically determine the mass of antibody addition.
I don’t see any trends in my antibody loading sweep. What happened?
The antibody loading ratios swept in this kit may not be optimum for your antibody or application. You may need to evaluate antibody loading ratios above or below the values investigated within this kit.
After I diluted my 50× reaction buffer, the pH was not within the acceptable range of 7.0–7.6. What happened?
This reaction buffer has an intentionally low buffering capacity. If diluted with water that has been exposed to the atmosphere for prolonged periods, the pH may no longer be neutral. Prepare a new dilution with fresh DI water.
What other factors can influence conjugation results?
If running under the correct pH conditions and the antibody incubation time has been optimized, confirm that EDC and sulfo-NHS has been stored properly and that it is solubilized just prior to conjugation. Sulfo-NHS should always be stored between 4–8°C, and EDC should be stored at –20°C or between 4–8°C. It is important to allow reagents to come to room temperature prior to opening the bottles to avoid condensation from the atmosphere as both EDC and sulfo-NHS are moisture-sensitive.
What other methods of functional testing can I perform if I do not have access to lateral flow strips?
Functional testing should closely mimic the end-use of your conjugate for accurate performance validation. For example: if the application of the conjugate is for a colorimetric ELISA, it is recommended that you coat wells of a 96-well plate with the appropriate antibody or protein to bind your conjugate. Applications may vary. If you have further questions regarding functional testing, please contact us at email@example.com, or call +1 (858) 565-4227.
Are the conjugates salt stable?
Unconjugated gold nanoparticles are not salt stable, but after successful conjugation, conjugates are generally stable in 1× PBS, and some customers have observed stability in much higher salt concentrations. If your application requires a high-salt environment, we recommend preparing your conjugate at different salt concentrations, and monitoring the conjugate stability by UV-Vis. A broadening in the peak and drop in optical density are indicative of instability.
Can I add more EDC and Sulfo-NHS to achieve better binding efficiency?
The amounts of EDC and sulfo-NHS in these protocols have been carefully determined to generate the best performance. A larger excess may cause the particles to aggregate, resulting in poor conjugation.
I am having trouble resuspending my conjugate after centrifugation.
The particles can sometimes form tough pellets that are difficult to resuspend. An ultra-sonicator is highly recommended for resuspension, but vigorous vortexing (~30 seconds on high setting) is usually enough to resuspend the pellet. Inspect the conjugate solution after resuspension to ensure there are no visible particulates.
Are your particles tolerant to detergents like Tween?
Stable conjugates optimized with this procedure are usually stable with most detergents and polymers commonly used in bioconjugation applications.
How can gold nanoshells increase sensitivity in lateral flow?
Our 150 nm gold nanoshells are 30× visibly brighter per particle than traditional 40 nm gold used in lateral flow. Because they have been engineered with a silica core, they are half as dense as a solid 150 nm gold particle and flow easily through a nitrocellulose membrane. It is important to note that for any OD per volume, there are about 30× fewer nanoshells by particle number, so conjugate volumes will need to be adjusted appropriately to maximize binding events. As a starting point to boost sensitivity, we recommend doubling either OD or conjugate volume per strip relative to the amount of 40 nm gold you would typically use. A full sweep of conjugate loading per strip is recommended.
Do you perform custom conjugations or assay development services?
Yes! Please contact us regarding our custom capabilities and collaboration efforts. We can be reached at firstname.lastname@example.org, or +1 (858) 565-4227.
I am having trouble interpreting my results.
For help interpreting your results, email us at email@example.com, or call +1 (858) 565-4227.
Can I use your BioReady materials for commercial applications?
Absolutely. Please contact us to discuss licensing and any applicable regulatory or compliance requirements for your intended commercial use. We can be reached at firstname.lastname@example.org, or +1 (858) 565-4227.
Why choose nanoComposix?
We are dedicated to providing superior products as well as offering the support our customers need to be successful with particle integration. We specialize in full-service lateral flow development and are excited to help our customers perform early-stage R&D or bring products to market.