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#28460673   2017/03/20 To Up

Proteomic characterization of canine seminal plasma.

The present study was conducted to identify the major proteome of the sperm-rich fraction and prostatic fraction of canine seminal plasma. Three semen samples from four healthy dogs were obtained by digital manipulation. The pre-sperm fraction, sperm-rich fraction and prostatic fraction were separated from each ejaculate. Immediately after sperm analysis, a protease inhibitor was added to the sperm-rich fraction and prostatic fraction, and the fractions were separately centrifuged and frozen at -80 °C. The samples were thawed, re-centrifuged, and the total protein concentration was determined. Samples were subjected to 1D SDS-PAGE and Coomassie-blue stained gels, were analyzed by Quantity One 1D Analysis Software. Bands detected in the gels were excised and proteins subjected to digestion with trypsin. Proteins were identified by nano-HPLC-MS and tools of bioinformatics. Tandem mass spectrometry allowed the detection of 268 proteins in the gels of sperm-rich fraction and prostatic fraction of canine ejaculate. A total of 251 proteins were common to the sperm-rich and prostatic fractions, while 17 proteins were present in the sperm-rich fraction and absent in the prostatic fraction. The intensity of the bands detected in range 1 and 2 represented 46.5% of all of the band intensities detected in the 1D gels for proteins of the sperm-rich fraction and 53.0% of all bands in the prostatic fraction. Arginine esterase and lactotransferrin precursor were the protein with the highest intensity observed in the both fractions. Among the proteins present only in the sperm-rich fraction, the proteins UPF0764 protein C16orf89 homolog and epididymal-specific lipocalin-9 were the most abundant. In conclusion, canine sperm-rich fraction and prostatic fraction express a very diverse set of proteins, with unique biochemical properties and functions. Moreover, although most proteins are common to both sperm-rich fraction and prostatic fraction, there are some exclusive proteins in sperm-rich fraction.
Annice Aquino-Cortez, Breno Queiroz Pinheiro, David Baruc Cruvinel Lima, Herlon Victor Rodrigues Silva, Antônio Cavalcante Mota-Filho, Jorge André Matias Martins, Paula Rodriguez-Villamil, Arlindo Alencar Moura, Lúcia Daniel Machado Silva

1529 related Products with: Proteomic characterization of canine seminal plasma.

500 ml100 ml500 ml0.05 mg100 ml0.05 mg200mg500 ml0.05 mg100mg100 ml500 ml

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#12375840   // To Up

Recovery of intact proteins from silver stained gels.

Silver stained proteins of a wide molecular weight (MW) range (20-116 kDa) were successfully recovered by both electroblot and electroelution. The recovery was demonstrated for nanogram loads of proteins separated by SDS-PAGE and visualized by silver staining methods compatible and incompatible with mass spectrometry (MS). It was shown that the alcohol/acid and glutaraldehyde fixation steps present in a number of staining procedures did not prevent recovery of intact proteins from gels. It was found that the recovery of intact proteins from silver stained gels was substantially increased upon pre-equilibration in a buffer containing the reducing agent, dithiothreitol (DTT). The effect of destaining on the recovery of silver stained proteins was also investigated. Comparable recovery of intact proteins within a wide MW range from silver stained gels with and without destaining step was demonstrated. Recovery of model proteins from gels visualized using silver staining method compatible with MS showed 52 to 76% yield of that from the unstained gel, depending upon method of the transfer. Comparison of the recovery of intact proteins from gels visualized using other staining procedures was also made. The above findings have implications as to the supposed irreversible nature of protein "fixation" inside polyacrylamide matrix, and confirm lack of binding of proteins in the gel to metal silver deposited on its surface. This method has the potential to be suitable for direct characterization of proteins by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) without additional purification steps.
Victor J Nesatyy, Neil W Ross

2123 related Products with: Recovery of intact proteins from silver stained gels.

1 mL1 mg1010.00 ug1mg1mg10.00 ug1mg1mg101mg25

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#2876884   // To Up

Human growth hormone (GH)-releasing factor stimulates and somatostatin inhibits the release of rat GH variants.

Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) was used for the analysis of proteins secreted by male rat pituitary cells in monolayer culture in the presence of 10 nM human GH-releasing factor (hGRF) or 30 nM somatostatin (SRIF) or in the absence of these factors. More than 300 medium proteins were reproducibly detected either by fluorographic autoradiography or by silver staining. Immunoreactivity of each protein was examined after 2D PAGE followed by Western blotting and immunostaining with affinity-purified antirat GH (rGH) antibody. While there was a cluster of immunoreactive spots in the GH dimer range (40,000-50,000 mol wt), at least 16 medium proteins of mol wt 22,000 or less were also stained. Among these 16 proteins the release of 15 was stimulated and the release of 14 was inhibited by hGRF and SRIF, respectively. On the other hand, there were 3 proteins of approximate mol wt 16,000 whose secretion was regulated in a coordinate manner as rGH by the hypothalamic factors but which did not cross-react with anti-rGH antibodies. The increase or decrease in the radioactivity of each protein spot obtained from media after pituitary cells were incubated with [35S]methionine and hypothalamic factors was analyzed statistically. A pulse-chase study suggested that at least 7 of the hormonally regulated proteins, including rGH, were synthesized very rapidly. Finally, the 2D PAGE analysis of cell-free translation products of messenger RNA derived from male rat anterior pituitaries revealed the presence of about 40 rGH-immunoreactive proteins which included pre-GH. These data suggest that there are multiple forms of rGH-variants or rGH-related proteins. The biological significance(s) of all the rGH immunoreactive proteins and of the GRF- and SRIF-regulated pituitary proteins remains unclear. It is evident that a number of these proteins are synthesized and released rapidly by pituitary cells in culture. Furthermore, the presence of multiple genes for these rGH-related proteins is suggested by the large family of immunoreactive gene products identified after cell-free translation of messenger RNA derived from the pituitary.
S Yokoya, H G Friesen

1549 related Products with: Human growth hormone (GH)-releasing factor stimulates and somatostatin inhibits the release of rat GH variants.

50 ug96T5 x 50 ug50 ug500 ìg20 ug102ug2ug10ug96 wells (1 kit)1 mg

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#7057116   // To Up

Identification of multiple subclasses of plasma low density lipoproteins in normal humans.

Density gradient ultracentrifugation of low density lipoproteins (LDL) from 12 normal subjects showed multiple, distinct isopycnic bands. Densitometric scanning of the gradient tubes revealed that each band could be assigned to one of four density intervals and that the boundaries of these intervals were consistent among all the subjects. Analytic ultracentrifuge flotation (S(f)(0)) rates were assigned to the four density intervals, and there was a strong correlation between peak S(f)(0) rate and peak isopycnic banding position (R(f)) of the LDL in the 12 subjects. The S(f)(0) value corresponding to the boundary between the two most buoyant LDL density subgroups was 7.5. This value is close to that previously demonstrated to define two LDL subdivisions (S(f)(0) 0-7 and S(f)(0) 7-12) that were discriminated by differing concentrations in men and women, and differing statistical relationships with levels of HDL and VLDL in a normal population. Further delineation of distinct subspecies of LDL was afforded by electrophoresis in 2-16% gradient polyacrylamide gels. Densitometric scans of protein-stained gels revealed multiple peaks, and particle diameters were assigned to these peaks using calibration markers. Particles of diameter >/= 280 A included both IDL and Lp(a), the latter defined by pre-beta mobility on agarose electrophoresis and density > 1.050 g/ml. LDL particles with diameters 220-272 A could be grouped into seven size intervals defined by modes in the distribution of gradient gel electrophoretic peaks in LDL from a group of 68 healthy men and women. Particle diameters of the major peaks in each of seven density subfractions decreased with increasing density of the fractions. However, particles within each of the size groups were distributed across a range of densities. Use of a lipid-staining procedure allowed identification of electrophoretic bands in whole plasma which corresponded to those seen in isolated LDL, eliminating the possibility that ultracentrifugation was responsible for formation of the subspecies detected by the gradient gel procedure. The application of density gradient ultracentrifugation and gradient gel electrophoresis provides a means of characterizing LDL from normal humans in terms of multiple distinct subpopulations which may also prove to have differing metabolic and pathologic properties.-Krauss, R. M., and D. J. Burke. Identification of multiple subclasses of plasma low density lipoproteins in normal humans.
R M Krauss, D J Burke

1602 related Products with: Identification of multiple subclasses of plasma low density lipoproteins in normal humans.

1 mg

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