Antigens, Receptors Labeling Reagent | A Novel Method for Fluorescent Labeling

Mechanism | Description | Protocol | Inquiry / Order

A new labeling method to the target protein on the cell surface
– Easy ID™ –

Mechanism of “Easy ID™”

Innovative labeling system by AGD chemistry (Affinity-Guided DMAP chemistry)

This system is the labeling method which can attach ** Acyl donor (thioester derivative – fluorescent dye modified) to the target protein with * DMAP linked to ligand as a catalyst.

Fluorescence-modified small molecules are covalently bonded to the target instead of ligands for receptors, antibodies (VHH, scFv, Fab) to antigens.

AGD chemistry mechanism: (a) recognition of protein by ligand (b) activation of acyl donor by DMAP (c) nucleophilic attack by substituent on amino acid of protein (d) elimination of ligand

“Analysis of Cell-surface Receptor Dynamics Through Covalent Labeling by Catalyst-tethered Antibody” †
Takahiro Hayashi, Yuki Yasueda, Tomonori Tamura, Yousuke Takaoka, Itaru Hamachi *
J. Am. Chem. Soc., 137, 5372-5380 (2015)

Difference in labeling position according to the length of DMAP linker used

Description

Product Name Quantity Price (USD)
Easy ID™ 5 units at standard protocol 355
Contents
*DMAP DMAP-Lys(DMAP)-Lys(DMAP)-[Pro]6-Gly-MDA-N-maleimido MW: 1521.8 0.1 mg
**Acyl donor 5(6)FAM-[g-Abu]-S-Ph MW: 553.1 0.1 mg

Protocol

◆Maleimide DMAP modification protocol

  1. Dissolve the desired protein, antibody, etc. in an appropriate buffer (pH 7 to 8 recommended) such as PBS.
    Finally prepare 50 μM, 300 μl of protein solution ①.
    (The protein solution can be 50 μl – 500 μl depending on the actual experiment scale.)
  2. Add 100 μl DMSO to this reagent to make 5 mM reagent stock ②.
    (It seems it would be better to add 50 μl or 100 μl DMSO in one bottle and make 5 mM stock.)
  3. Add 3 μl of equimolar reagent (DMSO stock solution ② above) to the protein of ① above and thoroughly mix the reaction solution by pipetting.
  4. Incubate at room temperature for 30 minutes to 60 minutes.
    (It is better to follow the reaction with MALDI MS. If the reaction does not proceed 100%, add 1 to 2 equivalent of reagent stock.)
  5. Purify the modified protein by gel filtration or the like.
Tips

During antigen labeling on the cell, the protein (antibody)-DMAP conjugate prepared above is made to have a final concentration of 1 μM.
Also, add Acyl donor to the cells (in the medium) to a final concentration of 8 μM. (See Hayashi et al., JACS (2015).)

Caution
  1. When the modification reaction does not proceed, it is recommended to reduce the protein of interest by DTT or TCEP. After reduction, remove surplus reducing agent.
  2. Incubation under nitrogen atmosphere is recommended to prevent re-oxidation of free cysteine.

◆Maleimide DMAP Modification Protocol (Reduced Version)

  1. Dissolve the desired protein, antibody, etc. in an appropriate buffer (pH 7 to 8 recommended) such as PBS.
    Finally make 50 μM, 300 μl protein solution.
    (Protein solution can be 50 ul – 500 ul depending on experiment scale.)
  2. Add 2-5 equivalents of DTT or TCEP to the protein.
    (You can use 5 mM reducing agent solution in the same buffer as protein.)
  3. Incubate at 4 °C for 1 hour.
  4. In case of DTT exclude extra DTT with spin column.
    (For TCEP go to the next step.)
  5. Add 100 μl DMSO to this reagent to make 5 mM reagent stock ②.
    (It seems it is better to add 50 μl or 100 μl DMSO in one bottle and make 5 mM stock.)
  6. Add 3 μl of equimolar reagent (DMSO stock solution ② above) to the reduced protein and mix the reaction solution thoroughly by pipetting.
  7. Incubate at room temperature for 30 minutes to 60 minutes.
    (It is better to follow the reaction with MALDI MS. If the reaction does not proceed 100%, add 1 to 2 equivalent of additional reagent stock.)
  8. Purify the modified protein by gel filtration or the like.
Caution

To prevent re-oxidation of free cysteine, incubation under nitrogen atmosphere is recommended.

Inquiry / Order

Please send e-mail to the address s.anaharabiologica.co.jp .