6294

Conjugation-Ready HRP Maleimide

$199.00

Conjugation-Ready HRP Maleimide, contains maleimide-activated HRP to enable the creation of stable protein-HRP conjugates. This product allows you to save time with pre-activated HRP, control HRP to target protein labeling ratios, and avoid HRP enzyme inactivation associated with periodate oxidation procedures. In addition, eliminate cross-linking events that lead to large protein aggregation and subsequent protein precipitation, which are common with glutaraldehyde techniques. Activated HRP-Maleimide is stable for long-term storage, unlike same-day-use alternatives.

Catalog # Size Quantity Price
6294 5.0 mg $199.00
Category:

Horseradish peroxidase (HRP) is commonly used as the signal reporter enzyme in ELISA-based assay formats. Older methods which utilized glutaraldehyde or periodate oxidation-derived Schiff base formation, have their drawbacks. Glutaraldehyde conjugation methods are difficult to control as they often lead to excessive cross-linking events that lead to protein precipitation. Periodate oxidation of the carbohydrate moiety of HRP can be a reasonably successful conjugation method. Unfortunately, the oxidation step necessary to form aldehydes on the HRP molecules can also lead to enzyme inactivation and this Schiff base linkage must be properly reduced and stabilized in order to form a permanent covalent bond between the HRP and the protein that is being labeled.

Utilization of maleimide- activated HRP (HRP-M) to facilitate the HRP conjugation process avoids the potential HRP enzyme inactivation associated with periodate oxidation procedures, while still controlling the HRP to target protein labeling ratios. This 2-step approach with pre-activated HRP eliminates the risk of uncontrolled cross-linking reaction events that lead to large protein aggregation and subsequent protein precipitation, which is a common occurrence when using glutaraldehyde techniques.

Required Materials:
• Protein (5 – 10 mg/mL in PBS, pH 7.4) to be labeled with HRP
• PBS, pH 7.3
• G-25 desalting column
• PBS containing 5 mM EDTA, pH 7.3
• PBS containing 10 mM EDTA, pH 7.8
• Traut’s Reagent (2-Iminothiolane)
• Spectrophotometer at A280 and A324
• Dithiodipyridine (DTP), 5 mM
• DiH2O
• Centrifuge concentrators
• L-Cysteine
• Iodoacetamide
• 1M boric acid, pH 9.7
• Dialysis supplies
Overview of protocol:
1. Protein Thiolation
2. Conjugation of HRP-M to Thiolated IgG
3. Termination of Maleimide Conjugation Reaction
4. Free/Reactive Thiol-Group Determination Using DTP (optional)
For detailed protocol, please see product documentation.

• Contains: 5.0 mg of activated HRP-Maleimide as lyophilized powder
• Molecular Weight: 40,000 plus 2-3 x 233 g/mole (HRP enzyme plus 2-3 conjugated maleimide conjugation linkers)
• Storage: -20°C