A kit to extract and fluorometrically determine the amount of mucin content in faces

Mucin Assay Kit


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Background

Mucins are a family of heavily glycosylated proteins, and main components of mucosa such as saliva, tear, gastric fluid enteric fluid. Basic configuration of mucin are a macromolecules linked ramiform sugar chain to peptide framework. The heterogeneous property of sugar chain makes them diversity, the molecules has various function, such as specific molecular recognition.

Some of the sugar chains recognize a specific protein derived from virus, bacteria was found. Mucins are positioned in mucosal barrier function in gut, stopping the translocation of pathogen and toxin into blood vessel beyond the intestinal wall. This kit is useful for determination of mucin content in feces.


Mucins are a family of high molecular (1000kda-10000kda) and heavily glycosylated protein. Mucin domains within the protein core are rich in threonine, serine and hydroxyproline, reducing end of sugar chain (GalNAc) are frequentry-linked to these amino acid by the post-translational O-glycosylation. This kit contains components to determine fecal mucin content.


Fecal mucin measurement can be used as an index of intestinal barrier function. This kit makes use of fluorometric detection to measure amount of mucin.
 


Step1: Extraction and partial purification of mucin from feces.
Step2: Determination of mucin O-glycosidically linked oligosaccharide chains is β-eliminated by diluted alkali, and reducing end of sugar chain is formed. Reducing carbohydrates are fluorescence-labeled at high temperature to produce intensity fluorescent condensate.

Structure of Mucin

Mucin and IgA protect the translocation of pathogen and toxin into blood vessel beyond the intestinal wall, as an intestinal barrier function.

Application

For determination of mucin content in feces

 

Feature and Advantages

Theory of measurement 

Prevent degradation of mucin by heat-denaturing the glucosidase in feces

Crude extract mucin

Decompose starch with Enzyme solution

Purify mucin with ethanol precipitation

Add Reagent@A to purified mucin and carry out heat treatment under alkaline conditions. (Reagent@A will react with the sugar reducing end of mucin and emit fluorescence after alkaline treatment)

Add Buffer C and measure the fluorescence (Excitation 336nm, Fluorescence 383nm)

Kit component

Buffer solution A (tablet): 3* for 100 mL
Buffer solution B : 1*25 mL
Buffer solution C : 1* 25 mL
Reagent A : 1* 1.0 mL
Reagent B : 2* 1.5 mL
Standard solution (N-acetylgalactosamine, 250 ug / mL ): *1 1.0 mL
Enzyme solution: 1* 1.5mL

Protocol

This product is designed for the measurement in fluorescence plate leaders (96 well plates).
When using Fluorescence plate leader, it can measure 100 samples.
When you measure with a light spectrophotometer, please use it with a microcell.

Preparation of Buffer A
Dissolve 1 tablet with 100ml of purified water.
Preparation of feces powder Feces should be freeze-dried and grounded in a mortar and stored at -20°C until use.

II.Measurement of fecal mucin 
1. Weigh 100 mg of feces powder into micro test tube (for 2ml), and add the 1mL of Buffer A, then mix the solution with bortex mixer for 30sec.


2. Heat the tubes at 95°C for 10 minutes to denature the glycosidase derived from bacteria.


3. Heat the tubes at 37°C for 90minutes to extract the mucins from the feces.


4. Centrifuge 20,000×g at 4°C for 15 minutes.


5. Transfer the 200uL of supernatant into another micro test tubes, and add 200ul of Buffer B, then mix the solution with bortex mixer


6. Add the 10uL of enzyme solution, mix together, then heat the tubes at 50°C for 20 minutes to resolve the dietary starch.


7. Cool down the tubes until room temperature, add 125ul of 99.5% ethanol, after mixing, settle the tubes at - 20°C for overnight.


8. Next day, centrifuge the tubes 20,000×g at 4°C for 10 minutes, remove the supernatant.


Add the 1ml of Buffer A to the precipitate, resolve the precipitate. (Sample solution)


9.Transfer the 20ul of sample solution and standard solution into the another micro test tube (for 500ul), add the 24ul of reagent mixture (mix together Reagent A and Reagent B, 1:5, just before use), after mixing, heat the tube up at 100°C for 30 minutes.


10.Cool down the tube until room temperature, add the 200ul of Buffer C, mix together with bortex mixer.


11. Transfer the 100ul of the solution into 96 well black plate wells, and measure the fluorescence using florescence plate reader set at a wavelength (ex:336nm em:383nm).


12. Create a standard curve by serial dilution as indicated in the below. Draw a smooth curve through these points to construct the calibration curve. Read the concentration for the samples from the calibration curve.


13. Calculation of mucin contents in the 1g of feces. Value measured at step (12) times 50 is mucin contents in the 1g of faces.

Working Calibrator: N-acetylglucosamine 250μg/ml
• Create a standard curve by serial dilution as indicated in the table below.
• The remaining undiluted Calibrator should be stored at 2-10°C.
• Diluted Calibrator is stable and should be stored at 2-10°C for 1 month.

 

 

 

 

This kit can be also used fresh or air drying feces.

Relevant Fields of Research

  • Probiotics
References

> Susumu Honda, Yoshikazu Matsuda, Masaye Takahashi, and Kazuaki Kakehi
Fluorimetric Determination of Reducing Carbohydrates with2-Cyanoacetamide and Application to Automated Analysis of Carbohydrates as Borate Complexes. (1980) Analytical Chemistry, Vol.52, No. 7
> Bovee-Oudenhoven IM, Termont DS, Heidt PJ, et al.: Increasing the intestinal resistance of rats to the invasive pathogen Salmonella enteritidis: additive effects of dietary lactulose and calcium. Gut 40: 497-504, 1997.
> Crowther RS, Wetmore RF: Fluorometric assay of O-linked glycoproteins by reaction with 2-cyanoacetamide. Anal Biochem 163: 170-174, 1987.
> Okazaki Y, Han Y, Kayahara M, Watanabe T, Arishige H, Kato N. Consumption of curcumin elevates fecal immunoglobulin A, an index of intestinal immune function, in rats fed a high-fat diet. J Nutr Sci Vitaminol (2010);56(1):68-71.
> Yukako Okazaki,Hiroyuki Tomotake, Kazuhisa Tsujimoto, Masahiro Sasaki,and Norihisa Kato. Consumption of a Resistant Protein, Sericin, Elevates Fecal Immunoglobulin A, Mucins, and Cecal Organic Acids in Rats Fed a High-Fat Diet. (2011) The Journal of Nutrition, 21, 10.3945/jn.111.144246.
> Zaki Utama & Yukako Okazaki & Hiroyuki Tomotake & Norihisa Kato, Tempe Consumption Modulates Fecal Secondary Bile Acids, Mucins, Immunoglobulin A, Enzyme Activities, and Cecal Microflora and Organic Acids in Rats. Foods Hum Nutr (2013) 68:177-183

Product List

Product Name Cat# Quantity Price

Fecal Mucin Assay Kit DataSheet ,

CSR-FFA-MU-K01E

1 KIT ¥42,000
$560

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.