Protein labeling protocol

Introduction

This Protein Labeling Kit is designed to label up to 1 mg of peptides or proteins such as antibodies with abberior dyes. Please read the entire protocol before you start.

Storage

The product is shipped at room temperature. After arrival, the product can be stored for up to one year at 4 °C. The product should be protected from direct light exposure.

Hazard and precautionary

This kit contains substances that are flammable and may be harmful in contact with the skin, eyes or respiratory tract. Appropriate protective clothing such as gloves should be worn. For further information, the safety data sheet is available on our webshop.

Protein labeling with abberior dyes

The procedure below has been successfully tested with our abberior dyes. The pure dye is activated at the carboxylic group and can then react with free amines on the protein under mild conditions to form a stable amide bond. This procedure has yielded consistent results in most instances. Due to the broad potential usage of the product and customer-supplied materials, further optimization for particular proteins or a specific degree of labeling (DOL) might be required. Please note that the functionality of peptides and proteins such as antibodies after coupling might change. The following protocol describes the labeling procedure for antibodies, Fab fragments, and Nanobodies. This Labeling Kit is not suitable for IgM proteins. Proteins smaller than 4 kDa can also be labeled with this kit, but purification using the included ultrafiltration system is not possible and must therefore be carried out individually. Working with these dyes in a well-lit room poses no risk, but direct sunlight should be avoided.

Required reagents; not provided

• Pure protein with a size of 4 kDa or higher (protein or protein solution must not contain BSA, serum, free amino acids, buffer systems with free primary amines)
• Reaction tubes for the coupling reaction
• Centrifuge with fixed angle rotor (40.45 °) for reaction vessels with a volume of up to 2 ml (speed of 14,000 xg)
• Assay and/or device for determining the protein concentration and the DOL (optional)

Labeling procedure

To avoid residues on/in the lid, the solutions or solids in the plastic tubes should be centrifuged. All steps are carried out at room temperature unless stated otherwise.

1. If necessary, dissolve the protein to be coupled in a suitable aqueous medium (not provided)
   
   Note: the medium must be free of BSA, serum, free amino acids, or molecules with free primary amines.
   Note: The concentration of the protein should be in around 1 mg/ml.
2. Add 10 % (v/v) of the BASE (white cap) to the protein solution from step 1. Gently mix by moving the liquid up and down with a pipette.
3. Add 7 µl of SOLVENT 1 (green cap) into the DYE tube (green cap). Dissolve the solid substance by pipetting up and down.
4. Add 500 µl of SOLVENT 2 (blue cap) into the ACTIVATOR tube (blue cap). Dissolve the solid substance by vortexing.
5. Add 3 µl of dissolved ACTIVATOR (blue cap) from step 4 into the tube containing the dissolved DYE (green cap) from step 3 and mix the solutions by vortexing.
6. Incubate the mixture from step 5 for 1 minute at room temperature.
   
   Note: The activated dye cannot be stored and should be used immediately after preparation.
7. Add the right amount (see examples in the table and use the formula below) of the activated dye from step 6 to the protein solution from step 2.
   
   

   	IgG	Fab fragment of IgG	Nanobody (VHH fragment)
   Molecular weight	∼150 kDa	∼50 kDa	∼15 kDa
   Maximal amount	1 mg	0.5 mg	0.23 mg

   
   IgG Antibody
   
   

   \(\text{Volume of activated dye solution (in µl)} =\frac{\text{1.5 × protein amount (µg)}}{\text{molecular weight of protein (kDa)}}\)

   
   
   Fab Fragment
   
   

   \(\text{Volume of activated dye solution (in µl)} =\frac{\text{1.0 × protein amount (µg)}}{\text{molecular weight of protein (kDa)}}\)

   
   
   Nanobodies, VHH-Fragment, single domain Antibody (sdAb)
   
   

   \(\text{Volume of activated dye solution (in µl)} =\frac{\text{0.65 × protein amount (µg)}}{\text{molecular weight of protein (kDa)}}\)

   
   
   Note: For other proteins with a different molecular weight, it may be necessary to adjust the amount of dye used.
8. Shortly flick and then swivel the protein-dye solution from step 7 for 30 minutes at room temperature.
9. Transfer up to 500 µl of the protein-dye solution from step 7 into the ultrafiltration vial, close the tube with the lid, and centrifuge the solution as described in the table below:
   
   

   Cut-Off	Time	Speed
   4 kDa	30 Minutes	14000 xg
   20 kDa	15 Minutes	14000 xg

   
   Note: The filter with a cut-off of 20 kDa can be used for antibodies or Fab fragments. For nanobodies (sdAb, VHH fragments) it is recommended to use the filter with a cut-off of 4 kDa.
   Note: Added and excess reagents such as DYE, ACTIVATOR, BASE, and SOLVENT are filtered out in this step.
   Note: Always use a counterweight for centrifugation. A 1.5 ml reaction tube of the same weight filled with water is sufficient for this.
10. Discard the filtrate in the filtrate collection tube (lower chamber).
11. Repeat steps 9 and 10 until the protein-dye solution from step 7 has been used up.
12. Add 100 µl of the PBS solution (transparent cap) or other aqueous medium suitable for the protein to the ultrafiltration vial and mix the residue with the added solution by carefully pipetting up and down avoiding the formation of bubbles and centrifuge the solution as described in the table below:
    
    

    Cut-Off	Time	Speed
    4 kDa	10 Minutes	14000 xg
    20 kDa	5 Minutes	14000 xg

    
    Note: Always use a counterweight for centrifugation. A 1.5 ml reaction tube of the same weight filled with water is sufficient for this.
    Note: Do not press on the filter with the pipette tip to avoid puncturing and destroying the filter.
13. Discard the filtrate in the filtrate collection tube (lower chamber).
14. Repeat steps 12 and 13 until the filtrate in the filtrate collection tube is almost colorless.
15. Take the ultrafiltration vial out of the filtration collection tube. Then, invert and place it into the fresh collection tube. Centrifuge the product from the membrane filter into the fresh collection tube for 2 minutes at 2,500 xg.
    
    Note: Always use a counterweight for centrifugation. A 1.5 ml reaction tube of the same weight filled with water is sufficient for this.
16. Determine the protein concentration of the final solution (e.g. using a Bradford assay or a Spectrophotometer with absorption at 280 nm).
    
    Note: The concentration of the conjugated protein should not be lower than 1 mg/ml.
    Optional: If the protein concentration is too high or needs to be adjusted to a specific value, simply dilute the protein solution with the PBS solution or other aqueous medium suitable for the protein.
    Optional: The average number of dye molecules per protein can be determined by the Degree of Labelling (DOL).) More info >
    Note: It may be necessary to optimize the DOL. For this purpose, it is recommended to adjust the volume of activated dye used.
    Optional: For a longer shelf life of the labeled protein, BSA (not included in the kit) at a final concentration of 2 mg/ml or sodium azide (not included in the kit) at a final concentration of 0.02 % to 0.2 % (v/v) can be added as a stabilizer. Only add these substances to the product if it is compatible with the labeled protein.
17. Transfer the protein solution into the storage container. Use the blank label to name the product and stick it on the storage container. Store the dye-labeled protein at optimal storage conditions for your protein.
    
    Note: The optimum storage conditions depend strongly on the protein. In most cases, however, 4 °C is sufficient for short-term storage (a few weeks) or -20 °C to -80 °C for several months.
    Optional: To avoid freeze-thaw cycles split the protein solution into smaller aliquots.