Chromogenic substrates

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11th December 2020

 

Apollo is thrilled to announce new distribution cooperation with Seramun Diagnostica GmBH, to expand our scope of products to our worldwide customers. Seramun Diagnostica GmBH strives on meeting the special requirements, ensuring the highest-level precision, keeping development times short and maintaining continual availability of quality-controlled products to its customers, following standardized procedures.

Initially, Apollo will distribute various SeramunBlau ELISA Chromogenic and Membrane substrates to coincide with our available reporter enzymes, Horseradish peroxidase(HRP) and Alkaline phophotase(AP). Each SeramunBlau substrate has been developed and tested in Seramun’s Berlin-based laboratory to obtain optimal detection with high sensitivity ranges to ensure a successful ELISA run. Available in short, moderate, and long reaction times, these substrates can perform at any incubation temperature with a considerable shelf life to ensure successful protein identification and analysis.

What is a Chromogenic substrate?

 

Chromogenic substrates are the simplest and most cost-effective method of protein analysis by colorimetric blotting detection. For specific protein detection, the chromogenic substrate will catalyse with a conjugated secondary antibody-producing a band or spot precipitant.[1] This coloured band can be seen on a nitrocellulose membrane with the naked eye and used to determine protein absorbance and concentrations on a standard curve.[2]

Chromogenic substrates are available depending on assay sensitivities, solubility, coloured precipitants, and reporter enzymes, most commonly, horseradish peroxidase(HRP) and alkaline phosphatase(AP). When dealing with HRP conjugated enzymes, researchers tend to use the highly sensitive substrate 3,3,5,5-tetramethylbenzidine(TMB).[3] TMB will produce a dark blue, water-soluble, precipitate when exposed to hydrogen peroxide and HRP at 650nm. Under further oxidation, the blue band will change to yellow at 450nm with the addition of sulphuric acid.[4]

When using the enzyme alkaline phosphatase(AP) another common substrate of choice is the highly sensitive and water-soluble, pNPP. During this hydrolyses reaction the product, p-nitrophenol and inorganic phosphate will produce a yellow precipitant at 405nm.[5]

Why this product could be useful in your research?

 

Each offered chromogenic substrate is a ready to use, high sensitivity solution for either horseradish peroxidase or alkaline phosphatase with variable reacting times. With each substrate dependent on a specific enzyme and method of detection it is very easy to customize these products to your specific research.  The offered variations of TMB substrates, from slow TMB to Automat TMB, also allows for a more customizable detection limit during an ELIZA run at any incubation temperature between 20°C and 37°C.[6]

The SeramunBlau chromogenic substrates can be used across various immunohistochemical applications including IHC tests, Western blots, and ELISA tests. And since the coloured precipitate products can be visualized on the nitrocellulose membrane without further equipment, it is easy to analyse data both quantitatively and qualitatively without additional equipment.

SeramunBlau Substrate Products:

 

Each new substrate item will be discounted 10% until the end of January 2021

BIL3125 SeramunBlau fast2: Fast reacting, ready to use, Horseradish peroxidase (HRP) substrate solution used to enable high sensitives in ELISA testing within 20minutes for in vitro systems

BIL3127 SeramunBlau slow2 50: Long reacting, ready to use, substrate solution used to enable high sensitives in ELISAs within 45minutes

BIL3128 SeramunBlau automat fast: Fast reacting, ready to use,  TMB/substrate solution used to enable high sensitivities in vitro detection ELISA systems within 20minutes.

BIL3129 SeramunBlau automat slow2: Moderate reacting, ready to use, TMB/ substrate solution used to enable high sensitives in ELISAs within 30minutes

BIL3130 SeramunGelb fast: pNPP/ substrate solution for Alkaline Phosphatase (AP) in immunological in vitro ELISA systems with a fast reaction time(20minutes).

1 https://www.cellsignal.co.uk/contents/_/elisa-(enzyme-linked-immunosorbent-assay)-detection-strategies/methods-of-elisa-detection

[2] https://www.mabtech.com/knowledge-center/tutorials-and-guidelines/enzymes-substrates/elisa-substrates#:~:text=The%20substrate%20is%20a%20crucial,be%20measured%20in%20a%20spectrophotometer.

[3] https://info.gbiosciences.com/blog/elisa-substrates-a-selection-guide

[4] https://media.cellsignal.com/pdf/7004.pdf

[5] https://www.sigmaaldrich.com/technical-documents/articles/biology/detection-methods.html

[6] https://www.seramun.com/en/products/substrates.html