Antibody Conjugation Troubleshooting
| Symptom | Issue | Recommendations |
|---|---|---|
| Little or no signal | Low antibody concentration |
To achieve the best results, it is important to closely adhere to the recommended antibody volume and concentration guidelines in the kit protocol. For example, compensating for a dilute antibody by adding a larger volume of antibody to the Lightning-Link® vial will dilute the Lightning-Link® reagents, lowering their ideal concentration. To increase conjugation efficiency, we recommend an antibody with a starting concentration greater than 0.5 mg/mL. If the starting concentration of your antibody is lower than 0.5 mg/mL, Antibody Concentration and Clean Up Kit can be used for quick and easy concentration. However, In addition to concentrating your antibody, this kit will concentrate larger proteins (BSA) if present in your antibody buffer, decreasing conjugation efficiency. To purify your antibody and remove BSA and other buffer additives that may interfere with the conjugation reaction, please visit our Support Kits and Services for Antibody Labeling page or email technical support for help choosing a kit. IMPORTANT NOTE: the suggested amounts (µg or mg) listed in the protocols refer to IgGs. If you would like to label an antibody or other molecule that is not an IgG, please contact technical support for advice. |
| Impure antibody |
Verify the purity of the antibody you attempted to conjugate. We recommend an antibody greater than 95% pure. The Lightning-Link® conjugation reaction targets available primary amine groups. If your antibody contains impurities, it may interfere with the conjugation reaction by competing for label with the target antibody. Unpurified antibody from ascites fluid, serum, or tissue culture supernatant will contain protein impurities. If you are using an antibody from one of these sources, then please visit our Support Kits and Services for Antibody Labeling page We offer a number of antibody purification kits. The most appropriate kit for use is sample-dependent; contact us at technical support for assistance with kit selection. IMPORTANT NOTE: Glycine and Tris reagents commonly used for elution and neutralization of samples are amine-containing and thus NOT compatible with the Lightning-Link® conjugation reaction. |
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| Antibody buffer/stabilizer(s)/additives(s) not compatible with Lightning-Link® |
Verify the formulation of the antibody you attempted to conjugate. If the buffer of your starting antibody contains BSA, Tris, azide, glycine, or other additives, then please visit our Support Kits and Services for Antibody Labeling page to learn more about antibody purification kits. Common antibody buffer additives can interfere with the conjugation reaction, particularly if present in combination. To perform a buffer exchange and to remove low molecular weight additives, we recommend the Antibody Concentration and Clean Up Kit. However, this kit utilizes a filter with a 10 kDa molecular weight cut-off and thus any proteins greater than 10 kDa in the antibody sample (such as BSA) cannot be removed using this method. In this case, please visit our Support Kits and Services for Antibody Labeling page or contact us at technical support for additional assistance with kit selection. |
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| Lightning-Link® pellet obtained moisture and collapsed/inactivated | The Lightning-Link pellet should appear full, fluffy (broken up is fine), and be easily resuspended. If the pellet shrinks and appears as a small ball which is difficult to resuspend, then the pellet may have obtained too much moisture. This is a rare problem, but may be caused by incorrect storage of the Lightning-Link® pellet (above -20 °C for long periods of time). | |
| Poor stability of the conjugate | Diluting the conjugate prior to storage | Storing the conjugate undiluted is recommended if possible; however, for HRP conjugates, if you wish to store at working concentrations (e.g. 1/10,000), we recommend use of LifeXtend™ HRP Conjugate Stabilizer/Diluent |
| Inherent instability of the antibody | Storing the conjugate undiluted is recommended if possible; however, for HRP conjugates, if you wish to store at working concentrations (e.g. 1/10,000), this can be done using LifeXtend™ HRP Conjugate Stabilizer/Diluent. The optimal storage conditions for each antibody are determined experimentally, using small aliquots of the conjugate. |
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| Azide present, inhibiting HRP | The preservative sodium azide inhibits HRP and should be avoided if using this label. | |
| PE conjugates stored at -20 °C | Conjugates containing PE should be stored at 4 °C | |
| Poor results with Lightning-Link® Biotin | Incorrect Lightning-Link® Biotin Type (A or B) used |
It is important to select the appropriate Lightning-Link® Biotin – A or B – for your application. Biotin A has been optimized particularly for performance; it should be used when the biotinylated antibody will undergo a wash step before coming into contact with streptavidin (or avidin). An example application is when biotinylated antibody is captured on an ELISA plate, followed by a wash step, and then streptavidin-HRP is applied. Biotin B has been specially developed to further minimize free biotin, and should be selected if the biotinylated antibody will NOT undergo a wash step before coming into contact with streptavidin (or avidin), e.g. if the biotinylated antibody will be captured on streptavidin beads. |
Related Resources:
Antibody Conjugation Illustrated Assay