Click-it® Plus Chemistry  

The copper concentrations typically used in traditional click chemistry reactions can affect fluorophores such as green fluorescent protein, mCherry, and R-phycoerythrin.

Click-iT® Plus EdU may be effectively employed in a low-copper reaction, enabling DNA synthesis–based cell proliferation assays that are compatible with multiplex imaging or flow cytometry experiments.

 

Copper sensitivity

Biomolecules exhibit varying sensitivity to copper concentration. The high copper concentration used to catalyze traditional click reactions maintains the fluorescence of APC, but not GFP. At medium copper concentrations, GFP and APC fluorescence is maintained, but phalloidin binding is lost (Table 1). With Click-iT® Plus reagents, the copper concentration can be optimized to preserve the signal in your fluorescent proteins and also drive the EdU click reaction.

Table 1. The effects of copper on fluorescence and phalloidin binding.

Copper concentration Maintained Lost
High
  • APC fluorescence
  • GFP fluorescence
  • Phalloidin binding
Medium
  • APC fluorescence
  • GFP fluorescence
  • Phalloidin binding
Low
  • APC fluorescence
  • GFP fluorescence
  • Phalloidin binding
 

Click-iT® Plus EdU vs. traditional click reactions

  • Faster than traditional BrdU
  • Brighter than traditional BrdU
  • Minimal cell disruption
  • Compatible with GFP multiplexing
  • Detection strategies for imaging and flow cytometry

When antibody-based BrdU detection is compared to click chemistry–based EdU detection, the HCl denaturation required for BrdU results in loss of signal from GFP. Traditional click chemistry generates a brighter signal than antibody-based BrdU, but the copper catalyst concentration results in loss of GFP signal. The low-copper reaction with Click-iT® Plus EdU results in bright EdU signals and retains the fluorescent signal from GFP (Figure 1).

Click-iT® Plus EdU
Figure 1. Cell proliferation assays using A375 melanoma cells expressing Erk2-GFP comparing traditional antibody-based BrDU (A), Click-iT® EdU (B), and Click-iT® Plus EdU (C).

Direct substitution with improved results

Click-iT® Plus EdU can substitute directly for your traditional Click-iT® EdU assay in either flow cytometry or imaging applications (Table 2).

Table 2. Replacing existing Click-iT® kits with Click-iT® Plus kits.

If you are using Replace with
Imaging applications
C10337 Click-iT® EdU Alexa Fluor® 488 Imaging Kit C10637 Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit
C10338 Click-iT® EdU Alexa Fluor® 555 Imaging Kit C10638 Click-iT® Plus EdU Alexa Fluor® 555 Imaging Kit
C10338 Click-iT® EdU Alexa Fluor® 594 Imaging Kit C10638 Click-iT® Plus EdU Alexa Fluor® 594 Imaging Kit
C10340 Click-iT® EdU Alexa Fluor® 647 Imaging Kit C10640 Click-iT® Plus EdU Alexa Fluor® 647 Imaging Kit
Flow cytometry applications
C10419 Click-iT® EdU Alexa Fluor® 647 Flow Cytometry Assay Kit, 50 tests C10634 Click-iT® Plus EdU Alexa Fluor® 647 Flow Cytometry Assay Kit, 50 tests
C10420 Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit, 50 tests C10632 Click-iT® Plus EdU Alexa Fluor® 488 Flow Cytometry Assay Kit, 50 tests
C10424 Click-iT® EdU Alexa Fluor® 647 Flow Cytometry Assay Kit, 100 tests C10635 Click-iT® Plus EdU Alexa Fluor® 647 Flow Cytometry Assay Kit, 100 tests
C10425 Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit, 100 tests C10633 Click-iT® Plus EdU Alexa Fluor® 488 Flow Cytometry Assay Kit, 100 tests