The application of proteomics is relevant to physiology, reproduction, immunology, muscle and lactational biology in animal science, altough its use is still limited. One of the greatest challenges of proteome analysis is the reproducible fractionation of the complex protein mixtures. The fractionation methods can increase the probability of bi...omarker protein discovery. The fractionation by liquid-phase isoelectric focusing is one of the prefractionation methods. As a result, protein fractions can be easily collected, pooled and refractionated. There is a lack in the knowledge of gel-based proteomic methods of egg as only a limited number of protocols can be found in the literature, thus sample purification and fractionation require a time consuming optimisation procedure. The aim of this study was to fractionate egg yolk and white proteins by isoelectric point in liquid phase.
In our study PCR-temporal temperature gelelectrophoresis (TTGE) and MeltINGENY bioinformatic program were used to analyse the mutations in the genes of melanocortin-1 receptor (MC1R) and pituitary adenylate-cyclase activating polypeptide (PACAP) in cattle. Amplification of target DNA by PCR was performed with GC-clamp primers and non-GC-clamp p...rimers in simplex PCR reactions. The fragments were separated by denaturing polyacrylamide gelelectrophoresis (denaturing agents: high temperature, urea) after PCR reactions.MC1R homozygous individuals were used for the reaction.
We concluded that MeltINGENY program makes the decision and detection system easier, and more simple as the melting profile of target sequence is determined by the software. In case of MC1R gene, PCR-TTGE method is appropriate for SNP detection, however PACAP gene polymorphism can not be identified by the method, because PACAP mutations are not included in melting domains, therefore PCR-TTGE cannot detect them.
Pituitary adenylate cyclase-activating polypeptide is a neuropeptide expressed primarily in the hypothalamus and in other tissues, which divergent and extensive physiological functions are proved. Large number of SNPs in PACAP are published involved SNP that is associated with phenotypic trait of cattle as well. Hungarian Grey, Hungarian Simmen...tal, Holstein, Charolais and Angus cattle breeds were involved in this study to search for polymorphism in exon 5 1–391 bp and G/A transition. Our results of PCR RFLP and PCR SSCP did not prove the occurrence neither G/A transition, nor any other SNP in cattle breeds involved.
The aims of the present study were to optimize a two dimensional polyacrylamide gel electrophoresis method to chicken liver proteome and to determine the changes of protein expression caused by selenium. Twelve broiler chicken were used in this experiment. The selenium intake was 0,2 mg/kg in the control group (6 chicken) and 4,25 mg/kg in the...experimental group (6 chicken). Using the optimized proteomic approach, we have succesfully separated 747 proteins in the experimental group and 741 proteins in the control group. We found six proteins, wich expressed only in the samples of experimental group. Further investigations need to determine these six proteins with mass spectrometry (MS) and look for the correlation between the physiological effects of selenium and the expression of these proteins.
Aim of our study was the optimization of a DNA method, that is appropriate for reliable, low cost identification of animal species in milk and dairy product (cheese) and to determine the ratio of species. Mitochondrial DNA was used in our work to analyse buffalo/cow milk mixtures contained different ratio of bovine milk such as 0.1%, 0.5%, 1%,...1.5%, 2%, 5%, 10%, 15% (v/v%). Buffalo cheese were produced using buffalo and cows milk (0%, 2%, 5%, 10%, 15% – v/v% cows milk in buffalo milk). In case of milk mixtures, using species specific primers, the PCR assay showed a 0.5 v/v% detection limit. Cattle, in the buffalo/cows milk 99.9/0.1 v/v% mixture, was not detectable. The identification of buffalo and cows DNA in cheese was successful. The intensity of eletroforetic PCR fragment indicated the increase of cow milk ratio in milk and cheese samples as well.
The aim of the present study was to determine marker proteins those are associated with functional longevity of dairy cattle. Holstein-Friesian cows were grouped based on their performance as follows: group 1) individuals with good longevity traits; group 2) short production life because of poor reproduction traits; group 3) short production li...fe with low milk yield. Twelve individuals were sampled in each group, blood and milk samples were collected from cows. Blood samples were analysed with two dimensional polyacrylamide gel electrophoresis (2D PAGE), MALDI TOF/TOF and nanoLCMS/MS. The milk samples were analysed with MALDI TOF/TOF and nanoLC-MS/MS. Using the optimized gel based proteomic approach,
we have succesfully separated 143 proteins in the group1, 139 proteins in the group2 and 136 proteins in the group3, but we could not find significant differences between groups in the expression pattern. Using MALDI TOF/TOF and nanoLC-IonTrap MS, we have found eleven protein sequences those were expressed only in the samples of good longevity group.