Antibody conjugation kit - Frequently asked questions (FAQs)

Frequently Asked Questions answered by our experts in Scientific Support

Questions

  1. What is the optimal starting concentration for the antibody?
  2. Do I need to purify the antibody before using the conjugation kit?
  3. Are antibody conjugation kits suitable for proteins and secondary antibodies?
  4. What buffers can be used?
  5. Which buffer additives can be used and what should be avoided?
  6. How do I remove additives from the antibody storage buffer?
  7. Does sodium azide cause interference?
  8. Is the antibody exposed to high pH?
  9. Do I need to desalt the final conjugated antibody?
  10. How do I store the conjugated antibody?
  11. Does BSA or gelatin affect the antibody labeling chemistry?
  12. Will my antibody form high molecular weight aggregates?
  13. What recovery can be expected?
  14. How do I filter out the free label from the conjugated antibody?
  15. What is the Antibody Concentration kit?
  16. What is the Antibody Purification kit?

 

1. What is the optimal starting concentration for the antibody?

Conjugation kits allow 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 conjugate to antibody. The kit sizes indicate the amount of IgG that can be labeled. Based on their molecular weights (e.g. 160kDa for an average IgG), 100-400μg of antibody can be added to 100μg of conjugate. Antibody concentrations of 1-4mg/ml give optimal results. (0.5mg/ml is the lowest we recommend you try) 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.

Note: These rules do not apply to PE and PE-tandem labels. For more details on the recommendations specific to these dyes, please consult the kit datasheet or protocol.

2. Do I need to purify the antibody before using the 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 conjugation or using purified antibodies. Ascites fluid, serum or hybridoma culture media can interfere with conjugation and should be avoided. View compatible and incompatible buffers in question 5.

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. As long as lysines free primary amines are present, secondary antibodies and proteins will be labeled with the conjugation kits. 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 would 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 and the best amount for your assay should be determined experimentally; our 3x10µg kits enable you to do this using small amounts of material and therefore at a low cost.

Note: The usual requirements will still apply - your target should be purified, in a suitable amine free buffer and not contain any additives such as Azide, BSA, Tris or Glycine.

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. The recommemnded pH for your buffer is between 6.5 and 8.5.

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 could interfere with the conjugation reaction.

Compatible additives:

  • up to 20mM Tris
  • up to 0.1% BSA (BSA up-to 1% will work but yield lower quality conjugates)
  • up to 0.1% gelatin
  • up to 0.1% sodium azide
  • PBS pH6.5-8.5
  • up to 50% glycerol
  • 0.15M sodium chloride
  • 50mM HEPES
  • 0.02M potassium phosphate
  • 0.001% Tween
  • Proclin 300
  • 5% Trehalose

Incompatibile additives:

  • Tris above 20mM
  • Urea
  • 50mM Imidazole
  • Glycine
  • Ethanolamine
  • DTT
  • Mercaptoethanol
  • Other thiols

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 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 a maximum concentration of 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. Additionally sodium azide should be avoided whenever possible and removed using one of our purification kits to ensure the highest possible quality conjugates.

8. 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.

9. 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.

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.). 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.

11. Does BSA or gelatin affect the antibody labeling chemistry?

Concentrations of up to 0.1% BSA and 0.1% gelatin have little effect on the conjugation chemistry.

12. Will my antibody form high molecular weight aggregates?

No. The 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?

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.

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, or to perform a buffer exchange.

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 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.


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