title: 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.
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, P.R. China, 712100.
authors.forename: Ruijie
authors.initials: R
authors.lastname: Hao
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, P.R. China, 712100.
authors.forename: Camus
authors.initials: C
authors.lastname: Adoligbe
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, P.R. China, 712100.
authors.forename: Bijie
authors.initials: B
authors.lastname: Jiang
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, P.R. China, 712100.
authors.forename: Xianlin
authors.initials: X
authors.lastname: Zhao
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, P.R. China, 712100.
authors.forename: Linsheng
authors.initials: L
authors.lastname: Gui
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, P.R. China, 712100.
authors.forename: Kaixing
authors.initials: K
authors.lastname: Qu
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, P.R. China, 712100.
authors.forename: Sen
authors.initials: S
authors.lastname: Wu
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, P.R. China, 712100; National Beef Cattle Improvement Center, Northwest A&F University, Yangling, Shaanxi, P.R. China, 712100.
authors.forename: Linsen
authors.initials: L
authors.lastname: Zan
mesh_terms: D000096:Acetone / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D011232:Chemical Precipitation; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D008055:Lipids / Q000737:chemistry; D018482:Muscle, Skeletal / Q000737:chemistry*; D009696:Nucleic Acids / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D020543:Proteome / Q000032:analysis; D040901:Proteomics; D000069466:Red Meat / Q000032:analysis*; D014238:Trichloroacetic Acid / Q000737:chemistry*
chemical_list: D008055:Lipids; D009696:Nucleic Acids; D011506:Proteins; D020543:Proteome; D000096:Acetone; D014238:Trichloroacetic Acid