Lightning-Link® Alkaline Phosphatase (AP)

The Expedeon Lightning-Link® Alkaline Phosphatase (AP) antibody labeling kit enables the direct conjugation of antibodies, proteins and peptides, or any other biomolecule with an available amine group, to Alkaline Phosphatase (AP) with only 30 seconds hands-on time and with 100% recovery of materials. The technology is fully scalable from 10ug to 1g or more and stringently QC tested for consistent high quality and excellent batch-to-batch reproducibility.

Alkaline Phosphatase (AP) Lightning-Link_process_diagram

Alkaline phosphatase (AP) is a hydrolase enzyme that is frequently conjugated to antibodies for use in immunoassays. PNPP is a popular colorimetric substrate for AP; the product of the reaction, p-nitrophenol, can be read at 405nM. Fluorogenic substrates (e.g. 4-methylumbelliferyl phosphate; MUP) may also be employed.

Please refer to our custom services for bulk orders or get in contact with us to know more about this product.

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Immunohistochemistry (IHC) data

Lightning-Link® Alkaline Phosphatase (AP)  IHC data

Figure 1. Dual staining of CD3 and CD20 with antibodies directly conjugated using Lightning-Link kits. Mouse anti human CD3 was conjugated with Alkaline Phosphatase, and mouse anti-human CD20 was conjugated using HRP. Both antibodies were used simultaneously to stain formalin-fixed, paraffin embedded human lymph node. CD20 positive cells are visualised suing DAB (Brown) and CD3 cells with Fast Red (Red). Direct labeling of monoclonal antibodies enables straightforward dual-colour staining in IHC, which is otherwise a highly complex and time-consuming technique.

 

ELISA data

Alkaline Phosphatase (AP) anti-Human IgG (Fc) Conjugates Analyzed by ELISA

Figure 2. Alkaline Phosphatase anti-Human IgG (Fc) Conjugates Analyzed by ELISA. Anti-human IgG monoclonal antibody was purchased pre-conjugated to alkaline phosphatase from a commercial supplier. The same antibody was purchased in un-conjugated form and then conjugated to alkaline phosphatase using a Lightning-Link® kit. The Lightning-Link® conjugate demonstrates enhanced titre and sensitivity.

Scalability Data

Lightning-Link® Alkaline Phosphatase scability data

Figure 3. Goat anti Rabbit labeled with Lightning-Link® Alkaline Phosphatase at 25ug, 250ug, 2.5mg and 25mg single reaction vials tested with Rabbit IgG in a sandwich ELISA. Detection: PNPP.

Stability Data

Lightning-Link® Alkaline Phosphatase stability data

Figure 4. Goat anti Rabbit labeled with 4 different batches of Lightning-Link® Alkaline Phosphatase at 100ug reaction vials tested with Rabbit IgG in a sandwich ELISA. Detection: PNPP.

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How to conjugate your antibody in one easy step

1. Which label should I use?

The choice of label for your antibody primarily depends upon the application:

Immunoassay Label
Western Blotting
  • Enzymes (usually HRP or Alkaline Phosphatase)
  • Fluorescent proteins and dyes
ELISA
  • Enzymes
  • Biotin/Streptavidin
Immunofluorescence
  • Fluorescent dyes
Immunohistochemistry
  • Enzymes
  • Biotin/Streptavidin
  • Fluorescent proteins and dyes
Flow Cytometry
  • Fluorescent proteins
  • Fluorescent dyes
  • Tandem dyes

Please see the table here for absorption/emission wavelengths of Lightning-Link® fluorescent dyes, proteins and tandems.

2. What is the difference between Lightning-Link® and Lightning-Link® Rapid?

Both our standard and rapid Lightning-Link® kits can generate conjugates in a hands-on time of typically 20-30 seconds for the entire procedure. However the conjugation and quenching incubation times are shorter for Lightning-Link® Rapid kits allowing the conjugates to be generated faster. The antibody considerations are exactly the same, as is the high quality of the final conjugate.

3. What can I label using Lightning-Link®?

Lightning-Link® technology works by targeting free amine groups on your target. It can be used to label antibodies, peptides, proteins and any other molecules with free amine groups.

For advice on labeling molecules other than antibodies please contact our technical support team.

4. How pure does my antibody need to be?

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.

5. Is my antibody in a suitable buffer?

For recommended buffer conditions please see the below table:

ll - b-pe - components correct

1 Please 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.

2 If 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.

6. 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.

7. 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.

8. 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.

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