Expedeon’s Lightning-Link® Alkaline Phosphatase AF (Animal Free) antibody labeling kit enables the direct conjugation of antibodies, proteins and peptides (or any other biomolecule with an available amine group) to alkaline phosphatase, with only 30 seconds hands-on time and with 100% recovery of materials. The technology is fully scalable from 10 µg to 1 g or more and stringently QC tested for consistent high quality and excellent batch to batch reproducibility.
Alkaline phosphatase is a metalloenzyme that catalyses the hydrolysis of phosphate monoesters. Alkaline phosphatase is frequently conjugated to antibodies for use in immunoassays based on colorimetric readouts. PNPP (para-Nitrophenylphosphate) is one of the most popular chromogenic substrates for alkaline phosphatase employed in ELISAs. Alkaline phosphatase catalyzes the hydrolysis of pNPP in inorganic phosphate and para-nitrophenol, which absorbance can be read at 405 nm. Fluorogenic substrates (e.g. 4-methylumbelliferyl phosphate; 4-MUP) may also be employed.
Alkaline phosphatase AF (Animal Free Origin) catalyses the same reaction of conventional alkaline phosphatase (EC 126.96.36.199) but it is produced in a microbial host, representing an alternative to bovine alkaline phosphatase, to support scientists working with assays where AF conditions are critical, without compromising on quality, stability and accuracy.
Additionally, paperwork and safety restrictions for transport and handling can be reduced as our AF alkaline phosphatase is provided BSA-free.
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Your antibody must be purified
because other molecules with free amine groups will interfere with the reaction
resulting in a poor quality conjugate.
A suitable method of purification
should be selected:
Affinity purification – this is the preferred method as it will always result in a purified sample containing only the antibody of interest.
Protein A or G purification of serum – this will result in a purified sample of all IgG’s contained in the serum. You will have a purified IgG fraction but only a small percentage of this fraction will be the IgG of interest. This IgG fraction will label with our kits, although adjustments in conjugate dilutions will be required.
Protein A or G purification of tissue culture supernatant or ascites fluid – this method will generate purified antibodies equivalent to affinity purification.
Other purification methods, such as ion exchange chromatography and ammonium sulphate precipitation, are low quality purification methods and should be avoided. Filtration using a 0.2/0.22 µm filter is a method of sterilization, not purification and is therefore not appropriate.
3. Is my antibody in a suitable buffer? For recommended buffer conditions please see the below table:
note that individually the concentrations shown should not affect the reaction.
However, in combination with additional compounds that are not recommended
above a certain concentration, the reaction may be affected.
intending to use this kit for immunohistochemistry, it is recommended that
there be no gelatin or BSA present.
3 It is important to note that sodium azide is a known inhibitor of HRP and should be avoided.
What can I do if my antibody formulation does not fit the requirements?
If your antibody requires purification or the buffer is not compatible with our kits, we have developed an AbSelect™ purification kit range that allows you to quickly and simply purify your antibody and is fully compatible with the Lightning-Link kits.
The appropriate kit to use depends on
your particular sample (species, buffer, contaminants, volume, etc.). We have
designed a handy flow chart to help you select a kit (view
here) but feel free to contact us if
you require further guidance. Please consult the individual kit protocols to
see the antibody amount/volume suitable for each kit.
If your antibody is already purified but its concentration is significantly lower than 1mg/ml, you can concentrate it using our Antibody Concentration and Clean Up Kit. This kit can also be used to remove low molecular weight contaminants such as azide, tris or glycine by carrying out a buffer exchange into the buffer supplied in the kit, which is fully compatible with Lightning-Link.
If your antibody contains BSA, you
can now use our BSA removal kit to
purify your antibody in a few simple steps. Please note this kit will also
enable you to concentrate your antibody.
NB: All the
AbSelect kits will ONLY work with antibodies. They will not purify other
molecules. The only exception is the concentration and clean up kit (861-0010),
which will work with other molecules greater than 10kD.
Do I need a wash or desalt step?
One of the advantages of the Lightning-Link technology compared to traditional labeling methods is that conjugate purification is not required. The Lightning-Link reactions are highly efficient, and the kits have been optimized so that, provided the protocols are followed, there should be only a very low amount of free label left at the end of the conjugation. This remaining free label would have its reactive ‘Lightning-Link’ groups blocked by the Quencher provided in the kit and is washed away during the wash step of your application.
How stable will my new conjugate be?
Upon dissolution of the Lightning-Link mixture with a solution of the antibody, proprietary chemicals in the mixture become activated. This results in the directional covalent bonding of the antibody to the label in a gentle and controlled process at near-neutral pH. The stability of your conjugate is dependent on your antibody and label of choice.
Most Lightning-Link conjugates can be stored for up to 18 months, undiluted at 4°C. The exception to this is tandem conjugates which are only stable for up to 3 months.
Please see here for
ordering & shipping information.
Lightning-Link® Alkaline Phosphatase AF (Animal Free) antibody labeling kit. Alkaline phosphatase AF (Animal Free Origin) catalyses the same reaction of conventional alkaline phosphatase (EC 188.8.131.52) but it is produced in a microbial host, representing an alternative to bovine alkaline phosphatase.