An Optimized Trichloroacetic Acid/Acetone Precipitation Method for Two-Dimensional Gel Electrophoresis Analysis of Qinchuan Cattle Longissimus Dorsi Muscle Containing High Proportion of Marbling

Abstract

Longissimus dorsi muscle (LD) proteomics provides a novel opportunity to reveal the molecular mechanism behind intramuscular fat deposition. Unfortunately, the vast amounts of lipids and nucleic acids in this tissue hampered LD proteomics analysis. Trichloroacetic acid (TCA)/acetone precipitation is a widely used method to remove contaminants from protein samples. However, the high speed centrifugation employed in this method produces hard precipitates, which restrict contaminant elimination and protein re-dissolution. To address the problem, the centrifugation precipitates were first grinded with a glass tissue grinder and then washed with 90% acetone (TCA/acetone-G-W) in the present study. According to our result, the treatment for solid precipitate facilitated non-protein contaminant removal and protein re-dissolution, ultimately improving two-dimensional gel electrophoresis (2-DE) analysis. Additionally, we also evaluated the effect of sample drying on 2-DE profile as well as protein yield. It was found that 30 min air-drying did not result in significant protein loss, but reduced horizontal streaking and smearing on 2-DE gel compared to 10 min. In summary, we developed an optimized TCA/acetone precipitation method for protein extraction of LD, in which the modifications improved the effectiveness of TCA/acetone method.

An effective placental cotyledons proteins extraction method for 2D gel electrophoresis

“…Similarly, Ruijie et al. (2015) had shown that protein lysates obtained directly from tissues that had been extracted using lysis buffers produced very dirty 2D proteomes. Therefore, this phenomenon might be due to the fact that proteins were not properly focused on the IPG strip.…”

“…Protein precipitation had been shown to improve proteome resolution via the removal of compounds that interfere with 2D‐GE . As mentioned earlier, protein pellet obtained from ic T/AHE was not completely dissolved even after thorough washing to remove TCA.…”

The Expression and Purification of Octa-Arginine Apoptin and Its Ability to Kill Cancer Cells

“…Gel concentration used are 15%. The protein from supernatant fraction of the cell culture precipitated by acetone precipitation method [6]. 250 µl of the supernatant mixed with 1 ml cold acetone, incubated for 2 h in-20 °C then centrifuged 12,000 rpm for 10 min.…”

“…On the other hand, the presence of related tags might also affect bioactivity of apoptin itself. It has been known that (his) 6 tag at C-terminus of heparin cofactor increases protein activity [3]. On the other hand, (his) 10 and (his) 7 Regardless of its modification, protein expression in a bacterial host might fail due to the differences of protein expression system between the original host and the new bacterial host.…”

Addition of urea and thiourea to electrophoresis sample buffer improves efficiency of protein extraction from TCA/acetone‐treated smooth muscle tissues for phos‐tag SDS‐PAGE

“…To avoid dephosphorylation during sample preparation, tissues or cell cultures are often fixed with cold trichloroacetic acid (TCA) or TCA/acetone . It has been reported, however, that re‐solubilization of proteins precipitated with TCA, is difficult in many cases, especially for two‐dimensional gel electrophoresis . It also sets a detection limit in SDS‐PAGE.…”