Questions
- What is the optimal starting concentration for the antibody?
- Do I need to purify the antibody before using the EasyLink conjugation kit?
- Are EasyLink conjugation kits suitable for proteins and secondary antibodies?
- What buffers can be used?
- Which buffer additives can be used and what should be avoided?
- How do I remove additives from the antibody storage buffer?
- Does sodium azide cause interference?
- Is the antibody exposed to high pH?
- Do I need to desalt the final conjugated antibody?
- How do I store the conjugated antibody?
- Does BSA or gelatin affect the antibody labeling chemistry?
- Will my antibody form high molecular weight aggregates?
- What recovery can be expected?
- How do I filter out the free label from the conjugated antibody?
- What is the Antibody Concentration kit?
- What is the Antibody Purification kit?
1. What is the optimal starting concentration for the antibody?
The EasyLink conjugation kit allows antibody labeling to be performed on a microgram to milligram scale. The amount of antibody should correspond to molar ratios between 1:4 and 1:1 of antibody to conjugate. Based on their molecular weights (e.g. 160kDa for the antibody versus 40kDa for the conjugate), 100-400μg of conjugate can be added to 100μg of antibody. Antibody concentrations of 0.5-5mg/ml give an optimal results. We recommend using 10μl, 100μl and 1ml of antibody solution with the 10μg, 100μg and 1mg kit formats, respectively. The antibody concentration for each conjugation kit has been optimised. Please refer to the relevant datasheet or protocol for the recommended antibody concentration.
2. Do I need to purify the antibody before using the EasyLink conjugation kit?
Yes. The antibody labeling chemistry involves free amine groups. Most proteins/peptides have lysine and/or alpha-amino groups, therefore, any protein/peptide present in the solution will also be labeled. We recommend purifying your antibodies before performing the EasyLink conjugation. Ascites fluid, serum or hybridoma culture media should be avoided. View compatible and incompatible buffers in question 5.
3. Are EasyLink conjugation kits suitable for proteins and secondary antibodies?
Yes. The labeling chemistry involves free 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. As long as lysines are present, secondary antibodies and proteins will be labeled with the EasyLink conjugation kits. However, they have not yet been specifically tested with secondary antibodies and proteins.
4. What buffers can be used?
We recommend using Hepes, MES, MOPS and phosphate-based buffers or any other amine-free buffer. Conjugation reactions can also be prepared in the presence of up to 20mM Tris buffer with almost no reduction in coupling efficiency. Once the reaction is complete, the conjugated antibody can be diluted in any buffer compatible with both label and antibody.
5. Which 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 on the labeling reaction. We recommend avoiding nucleophiles such as amino acids (e.g. glycine), blockers (e.g. ethanolamine) and thiols (DTT, mercaptoethanol) that might deactivate the chemical which covalently links the conjugate to the antibody.
Compatible additives:
- up to 20mM Tris
- up to 0.5% BSA
- up to 0.1% gelatin
- up to 0.1% sodium azide
- PBS pH7.4
- up to 50% glycerol
- 0.15M sodium chloride
- 50mM HEPES
- 0.02M potassium phosphate
- 0.001% Tween
- Proclin 300
- 5% Trehalose
Incompatibile additives:
- 60mM citrate + 150mM Tris pH7.8
- Urea
- 50mM Imidazole
- Glycine
- Ethanolamine
- DTT
- Mercaptoethanol
6. 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 EasyLink conjugation kit.
- The Antibody Concentration kit allows an easy concentration and reduction of azide, glycine and Tris.
- 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.
Learn more about concentration and purification kits in questions 15-16.
7. Does sodium azide cause interference?
Sodium azide can interfere during the conjugation reaction. We recommend conjugating an antibody in up to 0.1% sodium azide. Nevertheless, sodium azide is known as an irreversible inhibitor of HRP. We do not recommend performing a HRP conjugation in presence of sodium azide or adding sodium azide with your HRP conjugated antibody.
8. Is the antibody exposed to high pH?
EasyLink conjugation kits do not expose molecules to high pH. Labeling reactions are carried out at physiological pH.
9. Do I need to desalt the final conjugated antibody?
No. In WB, ELISA, IHC etc. you can use the conjugated antibody straight away.
10. 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.).
11. Does BSA or gelatin affect the antibody labeling chemistry?
Concentrations of up to 0.5% BSA and 0.1% gelatin have little effect on the conjugation chemistry.
12. Will my antibody form high molecular weight aggregates?
No. The EasyLink conjugation process does not enable antibody aggregation.
13. What recovery can be expected?
The entire antibody labeling reaction is contained within one tube. Recoveries are close to 100%.
14. How do I filter out the free label from the conjugated antibody?
EasyLink kits are designed to give a low level of free label at the end of the reaction. Thus no filtration steps are required.
15. What is the Antibody Concentration kit?
Our Antibody Concentration kit allows an easy concentration of antibodies and proteins. It can also be used to reduce the concentration of many unwanted contaminants such as azide, glycine, Tris by using a spin column.
16. What is the Antibody Purification kit?
Our Antibody Purification Kit quickly removes BSA, azide, glycine, Tris etc. and can also be used to purify antibodies from ascites or whole serum by binding the antibody to ProtA resine. The purification kit is not suitable for tissue culture supernatants because of their large volume that would need to be purified.
Back to EasyLink conjugation kit resources
For further information or help, contact our Scientific Support team.
