title: [Comparison of different methods of rat amniotic fluid sample preparation for 2-d electrophoresis].
abstract: OBJECTIVE
  To optimize the method for preparing samples for amniotic fluid proteomics study.
  
  
  METHODS
  Pregnant rats were sacrificed with an overdose of Chloral hydrate at E17. The fetuses and amniotic fluid were harvested. The samples were processed by three different methods including trichloroacetic acid (TCA)-acetone precipitation (protocol 1), TCA-acetone precipitation combined with an Albumin and IgG Removal Kit (protocol 2), and a Centrifugal Filter concentrating combined with an Albumin and IgG Removal Kit (protocol 3). The samples were run through a two-dimensional electrophoresis gel, stained and analyzed with a Image Master 6.0 software. Protein spots were identified with a LCQ Deca XP mass spectrometer.
  
  
  RESULTS
  The total numbers of protein spots for samples processed by protocol 1, 2 and 3 were 253 ± 28, 749 ± 32 and 782 ± 27, respectively. And there was a significant difference between protocol 1 has and other two methods. Those with MW > 50 kDa were 57± 14, 45 ± 13 and 41 ± 14, respectively. Protocol 2 differed significantly from protocol 3. Protein number of samples with MW < 50 kDa was 196± 29, 702± 35 and 735 ± 29, respectively. Again, protocol 1 has differed significantly from other two methods.
  
  
  CONCLUSIONS
  By removing albumin and IgG from the serum, low abundance proteins can be enriched with little loss of high abundance proteins. Centrifugal Filter concentrating combined with Albumin and IgG Removal Kit can be effectively applied for amniotic fluid proteomics study.
authors.affiliation: Department of Urologic Surgery, Shengjing Hospital, China Medical University, Shenyang, Liaoning 110004, P R China.
authors.forename: Li-ping
authors.initials: LP
authors.lastname: Shan
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.forename: Yang
authors.initials: Y
authors.lastname: Fan
authors.forename: Feng-hua
authors.initials: FH
authors.lastname: Zhou
authors.forename: Hui
authors.initials: H
authors.lastname: Gu
authors.forename: Zheng-wei
authors.initials: ZW
authors.lastname: Yuan
mesh_terms: D000653:Amniotic Fluid / Q000378:metabolism*; D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D011247:Pregnancy; D020543:Proteome; D040901:Proteomics / Q000379:methods; D051381:Rats
chemical_list: D020543:Proteome