Lightning-Link™ Frequently Asked Questions
Here you find answers to your Lightning-Link related questions.
Is the hands-on time really only 30 seconds?
Yes. Naturally you will need to familiarize yourself with the instructions before using Lightning-Link™ products for the first time, but after that your hands-on time should be no more than 30 seconds.
How long does the entire conjugation take?
The reaction will be complete after 3 hours at room temperature (20-25°C) with antibodies but a longer incubation time will not be detrimental. It is often extremely convenient to set up reactions overnight at room temperature and use the conjugate first thing the next morning.
Do I need to desalt the final conjugate?
Lightning-Link chemicals are designed to deactivate and are completely benign. In westerns, ELISA, IHC etc you can use the conjugate straight away.
How do I separate out free label?
You don’t need to. Lightning-Link conjugations are highly efficient and the conjugation kits are designed to give a low level of free label at the end of the reaction. Thus no separation step is required.
What buffers can be used?
Lightning-Link™ works with phosphate, Hepes, MES and MOPS and other amine-free buffers. Conjugates can also be prepared in the presence of 20mM Tris buffer with almost no reduction in coupling efficiency. Once the reaction is complete the conjugate can be diluted in any buffer that is compatible with both the label and the antibody.
What recovery can I expect?
The entire conjugation reaction is contained within one tube. Thus the usual losses on columns, dilution of samples, and further losses upon concentration simply dont occur. Recoveries are therefore as close to 100% as you are ever likely to achieve.
What functional groups do I need on my protein to use Lightning-Link™?
Lightning-Link requires amine groups on the molecule to be labeled. Most proteins or peptides have lysine and/or alpha-amino groups. All antibodies have multiple amine functions.
Can proteins other than antibodies be labeled?
Yes. While labeling of primary antibodies is a major application, the only requirement is that the protein or molecule to be labeled has amine functionality.
Is the label attached to my protein by a covalent bond?
Yes. Conjugates formed with Lightning-Link™ are perfectly stable.
Will my antibody couple to itself or form high molecular weight aggregates?
No. Although Lightning-Link™ is a one-step conjugation method it has no similarity to other simple methods (e.g. glutaraldehyde) which tend to give ill-defined aggregates. Moreover, direct coupling of antibody to label can be achieved without the troublesome desalting steps (e.g. following antibody thiolation or maleimide-activation of labels) that interrupt popular heterobifunctional methods. Lightning-Link™ combines the best of both worlds - speed and controlled coupling without desalting.
Is my protein exposed to high pH?
No. Unlike some conjugation methods Lightning-Link™ does not expose molecules to high pH. Conjugations are carried out at physiological pH.
Do I need to add preservatives?
Lightning-Link™ conjugates are just like any other conjugates. For long term storage at 4°C you may wish to add antimicrobial agents or stabilizers (e.g azide, BSA, glycerol, etc).
What buffer additives can be used and what should be avoided?
Additives such as salts (e.g NaCl), sugars (e.g. sucrose) and chelators (e.g. EDTA) have no effect. The main additives to avoid are nucleophiles that might deactivate the chemicals that conjugate the label to the protein of interest. Common nucleophiles used in laboratories include amino acids (e.g. glycine), blockers (e.g. ethanolamine) and thiols (DTT, mercaptoethanol).
Does sodium azide cause interference?
A slight reduction in efficiency has been observed in some head-to-head trials for samples with and without azide but for all practical purposes this is of little significance. The disadvantages of trying to purify small quantities of antibody (e.g. time required and losses of protein) mean that direct addition of the sample to Lightning-Link™ is the preferred method.
How scalable is the technology?
Scalability is one of the major advantages of Lightning-Link™. Rather than risk valuable antibody you can label a small quantity and be confident that the bulk quantity made later will have essentially identical properties. By avoiding desalting/dialysis steps the major source of losses and batch-to-batch variation in conjugation experiments is eliminated. Microscale optimisation and bulk production is a specialist service that we offer.
Are other quantities available for scale up?
The standard packs address the need for a technology that allows conjugations to be performed on a microgram scale to low milligram scale, but lyophilised material can be provided in any quantity with fast turnaround.
To discuss other quantities please email firstname.lastname@example.org
Do you offer a labeling service?
Yes. Please email email@example.com
How can I be kept updated on developments?
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