1. What is the optimal starting concentration for the antibody?
The amount of antibody should correspond to molar ratios between 1:4 and 1:1 of conjugate to antibody. The kit datasheets indicate the amount of IgG that can be labeled for each size of kit.
The ideal antibody concentration is 1mg/ml. 0.5 - 1 mg/ml can be used if the maximum antibody volume listed on the kit datasheet is not exceeded. Antibodies > 5mg/ml or < 0.5 mg/ml should be diluted /concentrated.
2. Do I need to purify the antibody or protein before using the conjugation kit?
Yes. The labeling chemistry involves free amine groups. Most proteins/peptides have lysine and/or alpha-amino groups, so any protein/peptide present in the solution will be labeled. We recommend only using purified antibody/protein with the conjugation kits. Antibodies in ascites fluid, serum or hybridoma culture media are not suitable for conjugation.
3. Are antibody conjugation kits suitable for proteins and secondary antibodies?
Yes. The labeling chemistry targets primary amines present in lysines and at the N-terminus of a protein. All antibodies have multiple free amine groups and most proteins have lysine and/or alpha-amino groups. The kits will work with antibodies, proteins, peptides and other biomolecules with available amine groups. Please bear in mind that the kits have been optimised for labeling IgGs. We recommend you adjust the amount of material you add to the label vial to allow for molecular weight difference. This should be done without changing the volume added to the vial, as this could affect the conjugation efficiency. As a rough guideline, we would recommend changing the amount of material proportionally to the size difference with IgGs. An average IgG is about 160kD, therefore for a target that is ½ the size of an antibody (about 80kD), add ½ as much to the vial. Please note this is only a guideline.
4. What buffers and buffer additives can be used?
The buffer requirements for the conjugation kits are listed below. Please contact us for questions on the requirements for the Gold conjugation kits and Oligo conjugation kits as they are slightly different to those given below.
Buffer should be pH 6.5-8.5.
Compatible buffer constituents
If a concentration is shown below, then the constituent should be no more than the concentration shown. If several constituents are close to the limit of acceptable concentration, then this can inhibit conjugation.
Tris1 50mM / 0.6%
BSA2 0.1%/1%
Glycerol 50%
Sodium azide3 0.1%
PBS
Potassium phosphate
Sodium chloride
HEPES
Sucrose
Sodium citrate
EDTA
Trehalose
EGTA
MES
MOPS
Borate buffer
Gelatin2 0.1%
1 Tris buffered saline is almost always ≤ 50 mM / 0.6%
2 1% BSA gives lower quality conjugates, BSA and gelatin can also interfere with the use of the conjugated antibody in tissue staining.
3 Sodium azide irreversibly inhibits HRP; antibodies with azide should be purified before using the HRP conjugation kit.
Incompatible buffer constituents
(in addition to any compatible constituents above the acceptable concentrations)
The buffer must not contain any constituents with primary amine groups or thiols (including the common constituents below):
Thimerosal
Proclin
Glycine
Arginine
Glutathione
DTT
Merthiolate
If a constituent of the buffer containing your antibody or protein is not listed above, please contact us.
5. How do I remove additives from the antibody storage buffer?
Our Antibody Concentration and Purification kits remove additives with ease and provide a ready-to-use antibody solution compatible with the conjugation kit.
The Antibody Concentration kit allows an easy concentration of antibodies and proteins, and reduction of the concentration of azide, glycine and Tris. It can also be used for buffer exchange.
The Antibody Purification Kit quickly removes BSA, glycine, Tris, azide etc. and can also be used to purify antibodies from ascites fluid or immune serum by binding the antibody to Protein A resin. The purification kit is not suitable for tissue culture supernatants because of their large volume that would need to be purified. For TCS and serum please see our TCS and serum specific antibody purification kits.
6. Is the antibody exposed to high pH?
The conjugation kits do not expose molecules to high pH. Labeling reactions are carried out at physiological pH.
7. Do I need to desalt the final conjugated antibody?
No. In WB, ELISA, IHC etc. and other applications where one would normally use secondary antibodies, you can use the conjugated antibody straight away.
8. How do I store the conjugated antibody?
For long term storage at 4°C we recommend adding antimicrobial agents and/or stabilizers (e.g. azide, BSA, glycerol, etc., and except that for HRP conjugates azide is not suitable). A new conjugate can be stored for 12-18 months at 4°C as long as the antibody will tolerate storage at 4°C. As the bond between the antibody and dye is covalent and very stable, it should not degrade, therefore at 4°C no additional preservatives are needed. The antibody is usually the least stable component of the conjugate so please check the antibody datasheet for storage recommendations. However, all conjugates can be stored in 50% glycerol at -20°C which allows them to remain stable for 2 years. Please bear in mind that APC and RPE conjugates should never be stored at -20°C on their own without glycerol. The dilution factor also has an effect. Storing the conjugate undiluted is recommended if possible.
9. Will my antibody form high molecular weight aggregates?
No. The conjugation process does not enable antibody aggregation.
10. What recovery can be expected?
The entire antibody labeling reaction is contained within one tube. Recoveries are close to 100%.
11. How do I filter out the free label from the conjugated antibody?
Conjugation kits are designed to give a low level of free label at the end of the reaction. Thus no filtration steps are required. Any remaining free label would have its reactive groups blocked by the Quencher provided in the kit, and would then be washed away during the relevant wash step of your application.
Learn more about conjugation kits
For further information or help, contact our scientific support team.