Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum

Summary

Bovine colostrum is both a primary source of nutrients and immunological support for the newborn calf. The understanding of the level of therapeutic proteins (lactoferrin and IgG) is important for the bovine colostrum dosing and standardization for human consumption.

Abstract

Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a good source of macro- and micronutrients, bioactive peptides, and growth factors. Bovine colostrum is also a potential source of nutrition and bioactive because of its rich protein content that includes immunoglobulin G (IgG) and lactoferrin. However, the level of lactoferrin and IgG in bovine colostrum changes markedly during the lactation period. Therefore, monitoring the concentration of IgG and lactoferrin for the use of bovine colostrum as a protein source is an important question to study. Methods in this article describe how to determine protein content, as well as specific concentrations of lactoferrin and IgG. These methods include the following steps: Isolation of bovine colostrum proteins, Determination of protein concentration via Bicinchoninic acid assay (BCA), Visualization of proteins via SDS-PAGE, Determination of lactoferrin, and IgG concentration using an ELISA Assay.

Introduction

Colostrum is the initial secretion of the mammary gland produced by mammals shortly after parturition. Colostrum is rich in macro- and micronutrients, antimicrobial peptides, and growth factors^1,2,3,4. The composition varies gradually over time through the transition to mature milk^5,6,7 but most significantly within 24 h after parturition⁸. The composition of colostrum is also influenced by maternal factors, including age, pa....

Protocol

Colostrum samples were collected for 6 days after calving in the noon over the period July-August, from 28 Holstein dairy cows from Uluova Milk Trading Company in Çanakkale, Turkey, and deep-frozen. The samples collected on the same day were pooled according to the day of each sample and analyzed for their total protein, lactoferrin, and IgG concentrations. All samples were assayed in duplicate.

1. Sample preparation

1. Mix 200 µL of bovine colostrum with 400 µL of dH₂O to obtain a diluted sample for the analysis. Dilute all the samples accordingly.
2. Centrifuge diluted and undilut....

Results

Following the protocol, the bovine colostrum samples were analyzed to determine protein, lactoferrin, and IgG concentration. The results of protein, lactoferrin, and IgG analyses of bovine colostrum are shown in Table 4.

Table 4: Concentration of protein, lactoferrin, and IgG of bovine colostrum.

Discussion

This study provides information about considerable changes in the protein, lactoferrin, and IgG concentrations in colostrum throughout the transition to mature milk. Detection of changes in the lactoferrin and IgG concentration was carried out by sandwich ELISA, and total protein concentration was analyzed by the BCA assay. Results indicate that early colostrum has the highest protein, lactoferrin, and IgG concentration, that subsequently decreased over the next 3 days. Accurate measurements of these proteins are relevan.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10X Running Buffer (Tris-Glycine-SDS)	ClearBand	TGS10	SDS-Page analysis
2-mercaptoethanol	gibco	31350-010	SDS-Page analysis
Acetic Acid GLACIAL	Isolab	901,013,2500	SDS-Page analysis
Bovine IgG ELISA Kit	Aviva Systems Biology	OKIA00005	Determination of IgG concentration
Bovine LF / LTF / Lactoferrin ELISA Kit	LSBio Lifespan Biosciences	LS-F4884	Determinaton of lactoferrin concentration
Coomassie Brillant Blue R 250	amresco	0472-25G	SDS-Page analysis
Hydrochloric Acid Fuming 37%	Isolab	932,103,2501	SDS-Page analysis
Isopropanol	Isolab	961,023,2500	SDS-Page analysis
Laemmli Sample Buffer (2X)	ClearBand	LSB-2x	SDS-Page analysis
Methanol	Isolab	947,046,2500	SDS-Page analysis
PageRuler Plus Prestained Protein Ladder 10 to 250	Thermo Scientific	26619	SDS-Page analysis
Pierce BCA Protein Assay Kit	Thermo Scientific	23225	Determination of protein concentration
Sodium dodecyl sulfate (SDS)	BioShop	SDS001.500	SDS-Page analysis
SureCast Acrylamide Solution 40% (w/v)	Invitrogen	HC2040	SDS-Page analysis
SureCast Ammonium persulfate (APS)	Thermo Scientific	17874	SDS-Page analysis
SureCast Tetramethylethylenediamine (TEMED)	Invitrogen	HC2006	SDS-Page analysis
TECAN Infinite M200 Plate Reader	Tecan	30035094	Measurement of absorbance
Tris base	BioShop	TRS001.1	SDS-Page analysis