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Cat. No.: HPG555-500 (for 500T)

Cat. No.: HPG555-2k (for 2000T)

Description

HPG Protein Synthesis Kit with Alexa Fluor 555, also known as HPG-555 Nascent Protein Assay Kit, is a reagent kit that enables simple, rapid, and highly sensitive detection of newly synthesized proteins. This is achieved through the incorporation of HPG (L-homopropargylglycine), a methionine analog, during protein synthesis and subsequent labeling of HPG with Alexa Fluor 555 via a click reaction.

This kit allows for in situ detection and quantification of nascent protein synthesis in cultured live cells. The detection process does not require radioactive isotopes (such as 35S-methionine) or antibodies, and it can be completed with a simple two-step reaction. Nascent proteins, also referred to as newly synthesized proteins, can be observed under a fluorescence microscope as bright red fluorescence, and this method is compatible with fluorescence microscopy, laser confocal microscopy, and fluorescence microplate readers, as well as high-content screening (HCS).

HPG (L-homopropargylglycine) is a methionine analog that can be incorporated into newly synthesized proteins during protein synthesis in place of methionine. The alkyne group on HPG can react covalently with small molecule azide probes (such as Azide Alexa Fluor 488, Azide Alexa Fluor 555, Azide Alexa Fluor 594, Azide Alexa Fluor 647, etc.) in the presence of a copper(I) catalyst to form a stable triazole ring. This reaction, known as the click reaction, is highly efficient and rapid. Through this click reaction, newly synthesized proteins are labeled with the appropriate fluorescent probes, allowing for detection using suitable fluorescence detection equipment.

Features

• This kit offers a simple reaction process and high detection sensitivity. It is based on an efficient click reaction that requires no special temperature conditions. With only a small amount of small molecule azide probes, HPG incorporated into newly synthesized proteins can be effectively labeled. The kit allows for the detection of protein synthesis in single cells as well as overall quantitative detection of protein synthesis.
• The kit is convenient to use and highly compatible. It requires only standard paraformaldehyde fixation and Triton X-100 permeabilization to allow the azide probes to enter the cells and undergo the click reaction effectively. This process does not affect cell morphology, does not produce unwanted byproducts, does not interfere with antibody-based immunofluorescence and immunohistochemistry assays, and does not affect DNA fluorescence staining (such as PI staining for cell cycle detection, DAPI, or Hoechst dyes for nuclear staining).
• The detection process is quick. The kit can detect newly synthesized proteins in just 1.5-2 hours.

Storage Conditions

Store at -20ºC, valid for one year. Azide 555 and Hoechst 33342 must be stored protected from light.

Precautions

• When preparing the Click Additive solution, properly aliquot and freeze. If a white precipitate forms after dissolving, invert the container several times until fully dissolved before use. If the solution turns brown, the component has become ineffective and should be discarded.
• If you need to use anisomycin as a control, you can order Anisomycin (JNK Activator).
• If you need more HPG, you can order L-Homopropargylglycine (HPG) (≥98%, BioReagent).
• Culture media and serum containing methionine will competitively affect the incorporation of HPG, thus influencing fluorescence intensity. It is recommended to use DMEM high glucose medium (without methionine) or RPMI 1640 medium (without methionine).
• As endogenous methionine cannot be removed from animal tissues, this kit is not recommended for detecting animal tissue samples.
• Since this product requires copper ion catalysis for the click reaction, please note the following compatibility issues and solutions. This product is fully compatible with organic dyes such as the Alexa Fluor® series, standard dyes, and Fluorescein (FITC), Allophycocyanin (APC), and APC-e-tandem dyes. For Qdot® nanocrystal probes, Horseradish peroxidase (HRP), R-phycoerythrin (R-PE), and R-PE-tandem dyes such as Alexa Fluor® 680-R-PE, reactions and detection should be carried out after the click reaction is complete. This product will affect the fluorescence of GFP, RFP, mCherry, and other fluorescent proteins. For fluorescent proteins such as Green Fluorescent Protein (GFP), TC-FlAsH™, and TC-ReAsH™ reagents, reactions and detection should be done before the click reaction. Since Phalloidin (Phallotoxin) is not compatible with the click reaction, it is recommended to use Tubulin-Tracker Red (Antibody-based Microtubule Red Fluorescent Probe) for cell microtubule detection.
• This product is for scientific research by professionals only and is not to be used for clinical diagnosis or treatment, food, or drugs. Do not store in a residential area.
• For your safety and health, wear a lab coat and disposable gloves during operation.