Establishing an optimized method for the separation of low and high abundance blood plasma proteins

Abstract

The study tested the efficiency and reproducibility of a method for optimal separation of low and high abundant proteins in blood plasma. Firstly, three methods for the separation and concentration of eluted (E: low abundance), or bound (B: high abundance) proteins were investigated: TCA protein precipitation, the ReadyPrep™ 2-D cleanup Kit and Vivaspin Turbo 4, 5 kDa ultrafiltration units. Secondly, the efficiency and reproducibility of a Seppro column or a ProteoExtract Albumin/IgG column were assessed by quantification of E and B proteins. Thirdly, the efficiency of two elution buffers, containing either 25% or 10% glycerol for elution of the bound protein, was assessed by measuring the remaining eluted volume and the final protein concentration. Compared to the samples treated with TCA protein precipitation and the ReadyPrep™ 2-D cleanup Kit, the E and B proteins concentrated by the Vivaspin4, 5 kDa ultrafiltration unit were separated well in both 1-D and 2-D gels. The depletion efficiency of abundant protein in the Seppro column was reduced after 15 cycles of sample processing and regeneration and the average ratio of E/(B + E) × 100% was 37 ± 11(%) with a poor sample reproducibility as shown by a high coefficient of variation (CV = 30%). However, when the ProteoExtract Albumin/IgG column was used, the ratio of E/(B + E) × 100% was 43 ± 3.1% (n = 6) and its CV was 7.1%, showing good reproducibility. Furthermore, the elution buffer containing 10% (w/v) glycerol increased the rate of B protein elution from the ProteoExtract Albumin/IgG column, and an appropriate protein concentration (3.5 µg/µl) for a 2-D gel assay could also be obtained when it was concentrated with Vivaspin Turbo 4, 5 kDa ultrafiltration unit. In conclusion, the ProteoExtract Albumin/IgG column shows good reproducibility of preparation of low and high abundance blood plasma proteins when using the elution buffer containing 10% (w/v) glycerol. The optimized method of preparation of low/high abundance plasma proteins was when plasma was eluted through a ProteoExtract Albumin/IgG removal column, the column was further washed with elution buffer containing 10% glycerol. The first and second elution containing the low and high abundance plasma proteins, respectively, were further concentrated using Vivaspin® Turbo 4, 5 kDa ultrafiltration units for 1 or 2-D gel electrophoresis.

Angiotensin-converting enzyme inhibitor activity of peptides derived from Kacang goat skin collagen through thermolysin hydrolysis

“…Protein separation by ultrafiltration showed that the value of protein content was largest at MW ≥5 kDa and that the low MW fraction after ultrafiltration was used for determining the bioactive peptide potency for ACE-1 testing. The higher protein content determined by ultrafiltration has been found at higher ultrafiltration size (more than 5 kDa); however, the protein obtained by enzymatic hydrolysis will be more efficacious [22]. Protein levels obtained were conducted by three repetitions and had significant (p<0.05) differences in terms of the ultrafiltration size.…”

Cibacron Blue F3GA ile modifiye polimerik mikroküreler ile RuBisCO adsorpsiyonu

“…CB-PHEMA mikroküreler ile saf RuBisCO çözeltilerinden adsorpsiyon sonrasında protein bantlarının yoğunluğunun azaldığı ve bant yoğunluklarındaki azalışın, artan CB-PHEMA miktarı ile arttığı belirlendi. [10,34,35]. Bitki proteom çalışmalarında, fotosentetik bitki dokularında biyobelirteç olarak kullanılabilecek fonksiyonel proteinlerin saptanmasında RuBisCO çok önemli bir sınırlayıcı proteindir [10].…”