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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 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™?
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 use
the customer
feedback form or email info@innovabiosciences.com.
Do
you offer a labeling service?
Yes. More details are available on the conjugation
services page.
How
can I be kept updated on developments?
To receive updates about new products just fill in
our customer
feedback form. Use this form to to make suggestions
for new labels, to express your interest in participating
in the development of novel products using Lightning-Link™
technology, or to ask questions not covered by these
FAQs.
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