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Invitrogen™ Click-iT™ Plus OPP Alexa Fluor™ 647 Protein Synthesis Assay Kit
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Catalog No. C10458
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Catalog No. C10458 Supplier Invitrogen™ Supplier No. C10458
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Description

Includes

Click-iT OPP Reagent, Alexa Fluor dye picolyl azide, Click-iT OPP Reaction Buffer, Copper Protectant, Click-iT Reaction Buffer Additive, Click-iT Reaction Rise Buffer and NuclearMask™ Blue Stain

Provides non-radioactive method for detection of protein synthesis using fluorescence microscopy or high-content imaging

The Click-iT™ Plus OPP Alexa Fluor™ 647 Protein Synthesis Assay Kit provides a fast, sensitive, and non-radioactive method for the detection of protein synthesis using fluorescence microscopy or high-content imaging. In this assay O-propargyl-puromycin (OPP) is efficiently incorporated into newly translated proteins in complete methionine-containing media and fluorescently labeled with a bright, photostable Alexa Fluor™ dye in a fast, highly-specific, and mild click reaction.

Features of the kit include:

• No media change required—works in complete, methionine-containing media, no need to remove cell media
• Multiplex-enabled—Click-iT™ Plus technology retains signal from GFP and binding of fluorescent-conjugated phalloidins
• Non-radioactive—an alternative to the traditional 35S-methionine methods
• Works in vivo—published results demonstrate use in vivo for determination of protein translation

The kit contains O-propargyl-puromycin (OPP), which is an alkyne analog of puromycin (also available separately), as well as Alexa Fluor™ 647 picolyl azide and all necessary reagents to perform the Click reaction. The OPP is fed to cultured cells and incorporated into proteins during active protein synthesis. Addition of the Alexa Fluor™ 647 picolyl azide and the Click reaction reagents leads to a chemoselective ligation, or 'click' reaction, between the picolyl azide dye and the OPP alkyne, allowing the modified proteins to be detected by imaged-based analysis.

The click reaction uses bioorthogonal (biologically unique) moieties to fluorescently label proliferating cells, helping to produce low backgrounds and high detection sensitivities. Because of the mild reaction conditions, Click-iT™ Plus assays detect protein translation events while enabling preservation of cell morphology, the binding of fluorescently-labeled phalloidin, and the fluorescent signal from GFP.

Unlike 35S-methionine, used in traditional methods, OPP is not an amino acid analog, so it can be added directly to cells in complete media or used to determine protein synthesis in vivo.

The kit contains all of the components needed to label and detect the incorporated OPP in newly translated proteins in samples of adherent cells. The kit includes sufficient reagents for the labeling of 25 18 mm × 18 mm coverslips using 1 mL of reaction buffer per test.

Specifications

For Use With (Application) Detection of protein synthesis using fluorescence microscopy or high-content imaging
Product Type Click-iT™ Plus OPP Alexa Fluor™ 647 Protein Synthesis Assay Kit
Quantity 1 Kit
Content And Storage This kit contains: Click-iT™ OPP Reagent, Alexa Fluor™ dye picolyl azide, Click-iT™ OPP Reaction Buffer, Copper Protectant, Click-iT™ Reaction Buffer Additive, Click-iT™ Reaction Rise Buffer, and NuclearMask™ Blue Stain. Store at 2° to 8°C. Desiccate and protect from light.
Reagent Type Nascent Protein Labeling Reagent
Product Line Click-iT
Label or Dye Alexa Fluor 647

Frequently Asked Questions (FAQs)

I will be performing a cell proliferation assay using Click-iT EdU kit. At what point can I stop overnight, or do I have to perform all the steps continuously?

One may store the sample after fixation overnight in PBS at 4oC. For longer storage (<1 week) , store in buffer with 1-2% formaldehyde or in formalin to limit microbial growth. If you use sodium azide as a microbial inhibitor, it must be completely removed prior to the Click-iT reaction.
Can the Click-iT Plus OPP Alexa Fluor 647 Protein Synthesis Assay Kit (Cat. No. C10458) be used for detection in flow cytometry?

The Click-iT OPP Assay Kits (Cat no. C10456, C10457 and C10458) can be used with any application that detects whole cells; therefore, the assay can be adapted for flow cytometry. You can follow the imaging protocol for labelling in the user guide. Briefly, you would need to spin the cells down (<2,000 rpm) to wash instead of covering the cells with the Click-iT Reaction Rinse Buffer as mentioned in step 5.6 on page 7 of the user guide. The picolyl azide dye concentration used in the click reaction has been optimized for imaging, which is less sensitive than flow. We recommend reducing this volume from 1/5th up to 1/10th the protocol volume for flow detection and making up the difference with 1X Reaction Buffer while keeping the volumes of the click reagents the same as in the protocol.