5 ways to achieve high quality conjugates
October 16th, 2014
Our Lightning-Link® antibody labeling kits for antibody and protein conjugation are quick and easy to use, however there are some important considerations to take into account when labeling your protein. Here are 5 ways to achieve high quality conjugates and optimal results from your experiment:
1. Select the best label for your application
There is a huge variety of antibodies and labels available commercially but which combination is best for your application?
One of the advantages of Lightning-Link® kits is that the technology can be used to conjugate any antibody to the wide variety of (over 40) labels in our range. The choice of label will usually depend on the experimental application. The table below provides a general overview of commonly used label types for several applications:
|Flow Cytometry/Immunofluorescence||Fluorescent dyes & proteins
|Western Blotting||Horseradish Peroxidase
Choosing the correct fluorescent label can be determined by the maximum absorbance, maximal emission and the extinction coefficient. We have 36 fluorescent dyes available which you can directly conjugate to your primary antibody, this table will help you choose which is most suitable for you.
2. Make sure your antibody is purified
Once you have selected the most suitable label for your conjugation it is important to ensure that your antibody is purified. This is because Lightning-Link® technology works by targeting amine groups on your biomolecule, therefore any free amine groups (i.e. if other proteins are present) will interfere with your reaction, resulting in poor quality conjugates. Affinity purification is preferred as it will always result in a purified sample containing only the antibody of interest.
Other methods of purification include:
– 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. Make sure your antibody is in a suitable buffer
Take a look a the below table for a summary of buffer considerations:
For recommended buffer conditions please see the below table:
|Amine free buffer (e.g. MES, MOPS, HEPES, PBS)||ü|
|Non-buffering salts (e.g. sodium chloride)||ü|
|Chelating agents (e.g. EDTA)||ü|
|Nucleophilic components (Primary amines e.g. amino acids or ethanolamine and thiols e.g. mercaptoethanol or DTT)||x|
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.
4. Check the amount and volume of antibody is correct
It is important to make sure you add the correct amount and volume of your antibody to the vial of lyophilised label to obtain conjugates with optimal performance. We recommend that you check the individual Lightning-Link® protocol for the amount and volume to add.
5. Store your new conjugate correctly
Post conjugation, it is important to store the newly labeled antibody correctly. For any new conjugate using a Lightning-Link® kit we recommend storage at 4°C, undiluted for 18 months. The exception to this is tandem conjugates which are only stable for up to 3 months.
The bond formed between the antibody and the dye is covalent and therefore very stable and should not degrade however it is important to check the storage conditions of the antibody to ensure they are compatible with these temperatures.
It is possible to store conjugates for longer at -20°C as long as the conjugate is stored with a cryoprotectant such as 50% glycerol. APC and RPE conjugates should never be stored at -20°C on their own without glycerol.
Please note the above are guidelines only – the best storage conditions may vary and should be determined experimentally.
Follow these 5 simple rules when labeling your biomolecule and you should achieve optimum results easily and efficiently!