746-0010 Lightning Link Conjugation Kit

1. Summary of features/benefits:

• Easy
  
• No solvents
  
• No powders to weigh out
  
• No desalting/dialysis
  
• High efficiency
  
• 100% recovery
  
• Freely scalable (custom pack sizes if required)

• 1 or 3 glass vials of Lightning-Link(TM) mix
  
• Modifier agent
  
• Quencher reagent
  
• Protocol

2. Protocol

2.1 Sample buffer
Ideally, the antibody to be labeled should be in 10-50mM amine-free buffer pH range 6.5 to 8.5. However, many buffers outside these limits of concentration and pH can be accommodated. Modest concentrations of Tris buffer are also tolerated. Appendix 1 gives further guidance on buffers and compatible additives.

2.2 Amount and volume of antibody
The amount of antibody used for labeling ideally should correspond to molar ratios between 4:1 and 1:1 Ab to HRP. Taking account of the molecular weights (160,000 versus 40,000), this means that for 100ug LL-HRP you need to add between 100-400ug of antibody. The volume of the antibody sample, ideally, should be up to 100ul (100ug pack size), up to 1ml (1mg pack size) and up to 5ml (5mg pack size). Antibody concentrations in the range 0.5-5mg/ml generally give optimal results, but concentrations and
2.3.1. Before you add antibody to the Lightning-Link(TM) mix, add 1ul of LL-Modifier reagent for each 10ul of antibody to be labeled. Mix gently.
2.3.2. Remove the screw cap from the vial of Lightning-Link(TM) mix and pipette the antibody sample (with added LL-Modifier) directly onto the lyophilised material. Resuspend gently by withdrawing and re-dispensing the liquid once or twice using a pipette.
2.3.3. Place the cap back on the vial and leave it standing for 3 hours in the dark at room temperature (20-25C). Alternatively, and often more conveniently, conjugations can be set up and left overnight, as the longer incubation time has no negative effect on the conjugate.
2.3.4. After incubating for 3 hours (or more), add 1ul of LL-Quencher FD reagent for every 10ul of antibody used. The conjugate can be used after 30 minutes.

2.4 Storage of conjugates
For any new conjugate, storage at 4C is recommended. A preservative may be desirable for long-term storage. Other storage conditions may also be satisfactory. The best conditions for any particular conjugate must be determined by experimentation.

Appendix 1. Compatibility of buffers and buffer additives. Amine-free buffers, including MES, MOPS, HEPES and phosphate are compatible if they are in the concentration range 10-50mM and have pH values in the range 6.5-8.5, as the addition of LL-Modifier provides the conditions necessary for efficient conjugation. Common non-buffering salts (e.g. sodium chloride), chelating agents (e.g. EDTA), and sugars have no effect on conjugation efficiency. Azide (0.02-0.1%) has little or no effect. Glycerol up to 50% has no effect.
If the amine-free buffer is relatively concentrated and outside the pH range 6.6-8.5 you may need to add more LL-Modifier for each 10ul of antibody. Excess LL-Modifier is provided so that you can check the pH of the buffer after the addition of the modifier. Ideally the pH should be around 7.3-7.6, though efficient conjugation occurs anywhere between pH 6.8 and 7.8.
Avoid buffer components that are nucleophilic, as these may react with Lightning-Link(TM) chemicals. Primary amines (e.g. amino acids, ethanolamine) and thiols (e.g. mercaptoethanol, DTT) fall within this class. (Note: Tris has little effect on conjugation efficiency as long as the concentration is 20mM or less).

3. FAQ's

Is the hands-on time really only 30 seconds?
Yes. Naturally you will need to familiarize yourself with the instructions before using Lightning-Link(TM) 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 (20-25C) 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?
In most cases no. 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?
If this is essential for your application, the considerations are the same as for any gel filtration experiment. A small sample volume (5% or less) relative to column volume is required. Since Lightning-Link(TM) conjugation processes do not lead to large increases in sample volume, the efficiency of separation on gel filtration columns is enhanced.

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 don't 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(TM)?
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(TM) are perfectly stable.

Will my antibody couple to itself or form high molecular weight aggregates?
No. Although Lightning-Link(TM) 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(TM) 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(TM) does not expose molecules to high pH. Conjugations are carried out at physiological pH.

Do I need to add preservatives?
Lightning-Link(TM) conjugates are just like any other conjugates. For long term storage at 4C you may wish to add antimicrobial agents or stabilizers (e.g azide, BSA, glycerol, etc).

What buffers can be used?
Lightning-Link(TM) 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 antibody used.

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(TM) is the preferred method.

How scalable is the technology?
Scalability is one of the major advantages of Lightning-Link(TM). 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.

Atto637 is a fluorescent label of new generation that emits in the red spectral region. It has a strong absorption at 630nm. It has a maximum emission at 630nm, with an extinction coefficient of 1.2 x105 cm-1M-1.
Atto637 can be used as a substitute dye for Alexa Fluor 633, BODIPY 630/650, DY-636, Cy5 and Light Cycler Red 640.

The Lightning-Link conjugation system represents a quantum leap forward in conjugation technology. It allows you to make antibody conjugates with minimal hands-on time - less than 30 seconds. Lightning-Link simplifies immunoassay techniques, such as western blotting, ELISA and immunohistochemistry, by eliminating secondary reagents and cutting the number of incubation and wash steps.

Despite its simplicity, Lightning-Link is a very sophisticated conjugation system in which the antibody is directionally coupled to the label (and not to itself) in a controlled fashion, creating high quality conjugates. As the procedure is not interrupted by desalting steps, trial conjugates can be prepared with microgram quantities of protein and then scaled up with ease. Lightning-Link eliminates problems associated with scale up; hands-on time is essentially independent of process scale. Consistency from batch to batch is easy to achieve, and recoveries approach 100%.