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. 2013 Nov 6:3:3145.
doi: 10.1038/srep03145.

Analysis of an ethanol precipitate from ileal digesta: evaluation of a method to determine mucin

Warren M Miner-Williams  1 , Paul J Moughan, Malcolm F Fuller
Affiliations

Analysis of an ethanol precipitate from ileal digesta: evaluation of a method to determine mucin

Warren M Miner-Williams et al. Sci Rep. .

Abstract

The precipitation of mucin using high concentrations of ethanol has been used by many researchers while others have questioned the validity of the technique. In this study, analysis of an ethanol precipitate, from the soluble fraction of ileal digesta from pigs was undertaken using molecular weight profiling and polyacrylamide gel electrophoresis. The precipitate contained 201 mg·g⁻¹ protein, 87% of which had a molecular weight >20 KDa. Polyacrylamide gel electrophoresis stained with Coomassie blue and periodic acid/Schiff, revealed that most glycoprotein had a molecular weight between 37-100 KDa. The molecular weight of glycoprotein in the precipitate was therefore lower than that of intact mucin. These observations indicated that the glycoprotein in the ethanol precipitate was significantly degraded. The large amount of protein and carbohydrate in the supernatant from ethanol precipitation indicated that the precipitation of glycoprotein was incomplete. As a method for determining the concentration of mucin in digesta, ethanol precipitation is unreliable.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Molecular weight profile (absorbance versus time) of the residue after ethanol precipitation of the 14,500 RCF digesta supernatant.
Figure 2
Figure 2. Molecular weight profile (absorbance versus time) of the supernatant after ethanol precipitation of the 14,500 RCF digesta supernatant.
Figure 3
Figure 3. Polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the residue from ethanol precipitation of the 14,500 RCF digesta supernatant using Coomassie Blue staining for protein.
* MW = Molecular weight (kDa) 1, 2 and 3 represent the volume of solubilised ethanol precipitate applied to the electrophoresis gel. 1 = 5 μL, 2 = 2.5 μL and 3 = 1 μL run against the marker in the right hand column.
Figure 4
Figure 4. Polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the residue from ethanol precipitation of the 14,500 RCF digesta supernatant using periodic acid Schiff staining for carbohydrate.
* MW = Molecular weight (kDa) 1, 2 and 3 represent the volume of solubilised ethanol precipitate applied to the electrophoresis gel. 1 = 5 μL, 2 = 2.5 μL and 3 = 1 μL.
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