SDS-page of magnetic isolated fraction from plasma of buffalo calves after depletion of high abundance proteins

Introduction

Proteins and peptides as a result of several biochemical reactions are secreted in blood indicating alteration in biochemical and physiological status of the animal. So, these can act as biomarkers of disease and physiological status of an animal. For identification of diseaseassociated biomarkers, proteome analytical techniques are being used. The use of current proteomic approaches in the search for potential diagnostic/prognostic indicators in the plasma of animals is limited by highly abundant proteins (Georgiou et al., 2001) [4] . Albumin and immune globulins constitute more than 60% of the total plasma proteins, making the hundreds or even thousands of other proteins difficult to detect by two-dimensional electrophoresis (2-DE). Depletion of abundant plasma proteins will improve the resolution of less abundant proteins that may prove to be informative disease markers. Over several decades, many approaches have been used to remove abundant plasma proteins, by methods based on the high affinity of some abundant proteins for certain textile dyes, such as Cibacron-Blue and several derivatives (Gianazza and Arnaud, 1982; Raymackers et al., 2000) [5,9] .

Materials and Methods

Blood collection and plasma separation

Ten apparently healthy buffalo calves of age up to six months were selected from buffalo farm, LUVAS, Hisar. Approximately 5 ml blood was collected from jugular vein using 10 ml disposable syringe aseptically for separation of plasma, the blood samples collected in centrifuge tube containing heparin were centrifuged at 3000 rpm for 10 minutes. The plasma samples were pooled and stored in aliquots till further analysis.

Sample preparation

100µl plasma was taken along with 100 µl diluted iron oxide magnetic particles in a micro centrifuge tube and vortexed at full speed of table top vortex at room temperature for 5 minutes to magnetically separate rejected proteins (cationic) in solution (not bound to iron oxide particles). Washing was done twice with ultrapure water on vortex @ full speed RT for 2 minutes and followed by magnetic separation. Anionic proteins bound to iron oxide particles were used for SDS-PAGE analysis.

Analytical procedure 12% resolving and 4% stacking gels were polymerized between two glass plates in a gel caster, with a comb inserted at the top of the stacking gel to create the sample wells. After the gel was polymerized, the inner chamber was filled with running buffer and the comb was removed. The samples to be analyzed and the Unstained Protein Molecular Weight Marker (Clontech Takara #3452, 200 kDa -6.5 kDa) were mixed with sample buffer in 1:1 ratio. The mixtures were heated at 95 °C for 5 minutes and then cooled before loading. A constant electric field (90 Volt) was applied across the gel, causing the negatively-charged proteins to migrate across the gel towards the positive (+) electrode (anode). Depending on their size, each protein was moved differently through the gel matrix.

Results

The gel was stained with coomassie blue dye. The molecular weights of the peptides were estimated by EZ Gel documentation system using Image lab software.

Discussion

Albumin constitutes 30 to 50% of the total plasma protein having molecular weight of 66.4 kD with three homologous domains in structure (Nakajou et al., 2003) [7] containing 67% of the protein as α -helix with no β -sheet (Curry et al.,

1998) [3] . Its concentration increases in dehydration while decreases in liver, kidney, gastrointestinal disease, acute phase response, malnutrition, blood and plasma loss. The acute phase response is an integral component of the innate immune response (Beutler, 2004) [1] forming the first reaction of the host to pathogens and tissue damage.

Monitoring the levels of the acute phase proteins (APP) can provide a way to assess the innate immune system's response to disease. But many acute phase proteins of diagnostic importance like α-1-fetoprotein, α-1-antithrombin III, α-2 -macroglobulin have molecular weights [2,6,8] . So monitoring the level and presence or absence of proteins of low abundance proteins can depict a picture in many disease conditions.

References

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