1% to 38% ( Fig. 1D; Supplemental Table 1). However, when all females were considered, acy3 expression and egg quality were not correlated ( Supplemental Figs. 2G and 3C). Two microarray features (20 K probe ID numbers 38561 and 48795) identified

as importin subunit alpha-8 (synonym: karyopherin alpha 7, kpna7) were > 2-fold higher expressed in fertilized eggs from the best quality female (2) compared with fertilized eggs from both of the lowest quality females (12 and 13) ( Table 2). qPCR showed that kpna7 transcript was detectable in the eggs of all females involved in the fertilized and unfertilized egg studies ( Figs. 3D and 4D). For both fertilized and unfertilized eggs, female 10 had the lowest kpna7 transcript expression (RQ of 1.0 for both studies; Supplemental Table 11 and Supplemental selleck kinase inhibitor Table 13). In fertilized eggs, the two females with the highest

kpna7 transcript expression were females 5 and 2 (RQ values of 64.1 and 27.8, respectively), while for unfertilized eggs females 9 and 2 (RQ values of 67.8 and 41.8, respectively) had the highest kpna7 transcript expression ( Supplemental Table 11 and Supplemental Table 13). It is interesting to note that females 2, 5, and 9 all had below average total mortality at 7 dpf (15.7%, 36.6%, and 28.5%, respectively, compared with an learn more average of 50.7%) ( Fig. 1C; Table 4). However, the association of high kpna7 expression and egg quality was not consistent. Some females with above PIK3C2G average egg

quality (e.g. females 3, 11, and 16) had relatively low kpna7 transcript expression ( Figs. 1C, 3D, and 4D). Further, when all females were considered, there was no correlation between kpna7 transcript expression and egg quality in either fertilized or unfertilized eggs ( Supplemental Figs. 2H and 3D). The hacd1 transcript was detectable in the fertilized and unfertilized egg from all females involved in the qPCR studies ( Figs. 3E and 4E). In the fertilized egg qPCR study, females 6 and 7 had the lowest hacd1 transcript expression (RQ of 1.0), and female 6 also had the lowest hacd1 expression in the unfertilized egg qPCR study ( Supplemental Table 11 and Supplemental Table 13). In both the fertilized egg and the unfertilized egg qPCR studies, the highest hacd1 transcript expression was measured for females 2, 5, and 9 (RQ values of 8.6, 8.4, and 11.0, respectively, for fertilized eggs; and RQ values of 8.2, 7.5, and 4.3, respectively, for unfertilized eggs) ( Figs. 3E and 4E; Supplemental Table 11 and Supplemental Table 13), all of which had below average total mortality at 7 dpf ( Fig. 1C; Table 4). As seen with kpna7, however, the association of high hacd1 expression with higher egg quality was not consistent, with some females with above average egg quality (e.g. females 3, 11, and 16) having relatively low hacd1 transcript expression ( Figs. 1C, 3E, and 4E).

Due to the sex differences in bone loss rate in later life [21], we additionally investigated genotypic effects separately in men and women; we found borderline evidence for a difference in the effects of rs9594759 (RANKL) on standing

balance by sex, with the effects only observed in women, and opposing directions of effect for rs3815148 (COG5) on standing balance, though we found no evidence for other differences. Genetic variants are generally not associated with typical confounders in observational epidemiology and, being fixed from conception, may be informative about the direction of causality [60]. We found no evidence of association between rs1801725 (CASR) and measures of anthropometry, physical activity levels or other demographic indicators. PLX-4720 solubility dmso Additionally, previous investigations of CASR polymorphisms have found evidence against associations with many traits including, vitamin D levels [61], osteoarthritis [19], osteoporosis [19] or hip BMD [19] and [20], as well as no [19] or only modest [20] associations with lumbar spine BMD; however, there is some evidence that their effects on BMD may be modified by birth-weight [62]. Although

a previous smaller study of 1252 females aged between 70 and 85 years found no associations between the SNP and either grip strength or timed up and go [17], our findings based on a larger number of individuals (n = 11,239) suggest that our observed association signaling pathway between rs1801725 and grip strength may indicate a causal role of raised serum calcium levels on poorer grip strength. The

association observed with grip strength but not with the three other physical capability phenotypes may be indicative Rucaparib of greater power due to the larger number of participants with available data with this trait. The inconsistent findings for the direction of effects for the BMD-raising alleles of the two SNPs considered and the associations observed for rs9594759 (RANKL) suggest further investigations are warranted in order to provide additional evidence for or against the causal role of BMD on physical capability. Previous smaller studies of older females (n = 421 [63] and 331 [64]) found no association between measures of physical performance, including grip strength, and SNP rs2234693, a variant in low LD (r2 = 0.04) with rs2941740 (ESR1). Our investigation was limited by the fact that we did not validate the genotypic effects of the SNPs on serum calcium, BMD or osteoarthritis in these studies. However, all of the SNPs chosen were robustly associated with their respective measures from large GWAS of individuals of European ancestry. The use of younger populations may help to elucidate whether associations are present at earlier stages of the life course.

Certain masses, foci, and areas of nonmass enhancement may be categorized as probably benign on baseline MR imaging. Elissa R. Price Magnetic resonance (MR) imaging is now an accepted component of standard breast-imaging practice. This article reviews the fundamentals of performing an MR imaging–guided biopsy using a grid localization system, and discusses many of the finer points and nuances of the procedure. Tips and tricks found useful at the authors’ institution are included, although multiple variations also exist. Performing effective and efficient MR imaging–guided biopsy depends both on deliberate

preparation (of the proceduralist, the patient, and the equipment) and on deliberate positioning (of the patient and find protocol the sampling device). Samantha L. Heller, Ozvaldo Hernandez, and Linda Moy Breast magnetic resonance (MR) imaging is increasingly performed for a variety of indications, most commonly with the goal of detecting breast cancer. Percutaneous biopsy (usually under MR guidance or ultrasound if there is a correlating finding) is commonly used to evaluate suspicious imaging findings detected on MR imaging with the goal of identifying malignancy. It is important to be familiar with the characteristics and

management of high-risk lesions detected or biopsied under MR guidance. This review focuses on the appearance of a variety of breast lesions detected on MR imaging that require excision with focus on pathologic correlation. Savannah C. Partridge Carnitine palmitoyltransferase II and Elizabeth S. McDonald Diffusion-weighted magnetic resonance (MR) imaging (DWI) has shown promise for improving the positive Galunisertib nmr predictive value of breast MR imaging for detection of breast cancer, evaluating tumor response to neoadjuvant chemotherapy, and as a noncontrast alternative to MR imaging in screening for breast cancer. However, data quality varies widely. Before implementing DWI into clinical practice, one must understand the pertinent technical considerations and current evidence regarding clinical applications of breast DWI. This

article provides an overview of basic principles of DWI, optimization of breast DWI protocols, imaging features of benign and malignant breast lesions, promising clinical applications, and potential future directions. Patrick J. Bolan In vivo magnetic resonance spectroscopy (MRS) of the breast can be used to measure the level of choline-containing compounds, which is a biomarker of malignancy. In the diagnostic setting, MRS can provide high specificity for distinguishing benign from malignant lesions. MRS also can be used as an early response indicator in patients undergoing neoadjuvant chemotherapy. This article describes the acquisition and analysis methods used for measuring total choline levels in the breast using MRS, reviews the findings from clinical studies of diagnosis and treatment response, and discusses problems, limitations, and future developments for this promising clinical technology.

Ethical approvals for the use of clinical notes and for the research study were obtained from The

Gambian Government/MRC Laboratories Joint Ethics Committee. Written informed consent was obtained from the family. The father did not participate in the study. A detailed clinical assessment was conducted to identify the presence of any clinical signs and symptoms of rickets including; enlarged wrists or ankles, leg pain, difficulty walking and bow-leg or windswept deformity, and to discount other diseases associated with bone deformities. Bilateral radiographs were taken of knees and wrists of the affected children and were scored by a consultant paediatrician (JMP) using a 10-point scoring system developed by Thacher et al. [6]. Standard anthropometry was conducted which included weight (wt) and standing height (ht). An overnight-fasted, 2 h urine (u) sample was collected between BI 6727 manufacturer the hours of 07.00 and 09.00. Acidified (HCl 10 μL/mL, laboratory reagent grade SD 1.18, Fisher Scientific UK Ltd., Loughborough, UK) urine aliquots were stored at − 20 °C DAPT mouse and then later transported frozen on dry ice to MRC Human Nutrition Research (HNR), Cambridge, UK for analysis. A fasting, venous blood sample was collected, in the middle of the 2 h urine collection, transferred to lithium heparin (LiHep) and EDTA-coated tubes, plasma separated by centrifugation at

4 °C and frozen at − 20 °C, and later transported frozen on dry ice to MRC HNR, where the plasma samples and the blood cell pellets were stored at − 80 °C until analysis. The plasma samples were

analysed for markers of vitamin D, Ca and P metabolism using commercially-available methods according to the manufacturers’ instructions: intact PTH (Immunoradiometric assay; DiaSorin Ltd., Berks, UK), FGF23 (C-terminal ELISA; Immutopics Inc., CA, USA), 25OHD and 1,25(OH)2D (radioimmunoassay DiaSorin, Vasopressin Receptor MN, USA and IDS, Tyne and Wear, UK respectively). The following colorimetric methods (Cobras Fara, Roche Products Ltd, UK and Konelab™ Analyser 20i, Finland) were used to determine plasma analytes: total calcium (TCa) by methylthymol blue (Roche Unit-Kit II) and arsenazo III (Konelab™ 981367); P, ammonium molybdate (Roche Unit-Kit II and Konelab™ 981890); and total alkaline phosphatase (TALP), p-nitrophenyl phosphate at 37 °C (Roche Alp MPR2 and Konelab™ 981832). For FGF23, > 125 RU/mL was used as an upper-limit cut-off of normality. Acidified urine was used to determine urinary (u) uCa and uP employing the same colorimetric methods as for plasma and uCr was determined using the Jaffe method (Konelab™ 981832). uCa excretion was expressed as a molar ratio with uCr. Tubular maximal reabsorption of phosphate (TmP:GFR) (mmol/L) was determined in the following way: Tubular reabsorption of phosphate (TRP) = 1 − (uP/P) × (Cr/uCr), if TRP < 0.86 then TmP:GFR = TRP × P mmol/L, if TRP > 0.86 then TmP:GFR = (0.3 × TRP/1 − (0.8 × TRP)) × P mmol/L [7].

Attenuation of vaccines An attenuated vaccine contains an infectious, but less virulent, pathogen that induces a mild form of disease. Attenuated vaccines typically stimulate strong, durable antibody- and cell-mediated immune responses. An attenuated vaccine has the disadvantage of potentially being associated with a small risk of vaccine-related disease, especially

in individuals with underlying impairment of immune function. Furthermore, for some attenuated vaccines, there are safety concerns about the potential for reversion from the attenuated form back to a virulent one. Almost in parallel to attenuated pathogens, researchers started working on inactivated pathogens. These were initially developed for veterinary applications, based on the observation AZD5363 molecular weight that inactivated pathogens maintained the ability to induce protection. The first inactivated selleck screening library vaccines developed for human use were against

typhoid, cholera and plague. How inactivated vaccines were discovered Formaldehyde was used in Gaston Ramon’s laboratory to clean and sterilise test tubes and glass flasks. One of the flasks used for toxin preparation was not thoroughly rinsed and the remaining formaldehyde was sufficient to inactivate bacterial toxins (1924). This observation appears to have originated the use of formaldehyde inactivation in vaccines. Typhoid fever, a disease spread easily under primitive sanitary conditions and by chronic carriers (Figure 1.7), was highly feared at the beginning of the 19th century due to its high case-fatality rate of up to 20%. To protect troops against typhoid fever, the military initiated the development of a whole cell, inactivated bacterial vaccine. Typhoid vaccination was first tested in 1896 in 2835 volunteers of the Indian army (Levine, 2008). Consequently, the army decided to vaccinate soldiers sent to the Boer War. The vaccine caused some adverse events but a committee reviewed the available data and concluded

that the benefits from prevention of the disease outweighed the risks from vaccination; this may be Florfenicol the first example of an assessment of the risks and benefits of vaccination. There are, however, some disadvantages associated with inactivated whole pathogen vaccines. Multiple doses are generally needed to provide sufficient stimulation of the immune system and booster doses may be needed to induce or maintain persistent immune responses. While live, attenuated and inactivated pathogen vaccines were effective, in the early days of vaccine manufacturing there were many issues including contamination, potency and quality of pathogen production, and lack of standardised harvesting processes.

Notably, recent innovation in ICR-cell technology potentially provides similar performance at a lower magnetic field strength [33]. The statistical analysis of profiles generated from a clinical cohort of samples allowed the discrimination between healthy individuals and PC patients with sensitivity and specificity comparable with those reported by other authors using MALDI-TOF MS. A total of 273 serum samples was processed and mass analyzed within a time frame of 24 h and the high quality of the data both facilitated the interpretation and evaluation of the generated profiles. These ultrahigh resolution mass spectra represent selleck kinase inhibitor a “next-generation” of MS-based peptidome profiles and provide a new

tool for a more detailed description of the high-abundant proteins in clinical serum sample cohorts aiming for new diagnostic leads. “
“Breast cancer, the most frequent cancer entity among women, is nowadays recognized as a heterogeneous disease in terms of tumor morphology as well as at the molecular selleck chemical level [[1], [2] and [3]]. Treatment of breast cancer patients with similar clinicopathologic features can result in different outcomes regarding disease progression and survival. Over the last few years, gene expression profiling has provided insights into molecular mechanisms

associated with observed heterogeneous clinical outcome [4]. The seminal work of Sorlie and Perou identified intrinsic molecular subtypes, termed luminal A, luminal B, basal-like, and HER2-enriched, with unique biological Celecoxib and prognostic features [5]. The largest group of breast cancer patients suffers from luminal breast cancer with overexpression of hormone receptors as molecular hallmark. Luminal breast cancer comprises patients of the luminal A subtype with good prognosis whereas patients of the luminal B subtype are at a higher risk to suffer from recurrence [6]. Treatment of patients in these two groups is fundamentally different,

with patients at higher recurrence risk requiring chemo-endocrine treatment, whereas others do not benefit from chemotherapy. Hence, to avoid over- or under-treatment of patients with luminal breast cancer, tools allowing a clear-cut distinction of low and high risk are required. Although different approaches employing gene expression signatures or protein-based assays were introduced [[7], [8], [9], [10] and [11]], a robust assessment of the recurrence risk in luminal breast cancer has remained a challenge. To differentiate between low and high risk tumors, proliferation rate has emerged as a prominent feature, mainly supported by gene expression profiling data [4,12]. This is in line with information provided by histologic grade which is beside age, tumor size, and lymph node status a well-established independent prognostic factor, combining information on tumor proliferation and differentiation status.

Before nucleic acid extraction, the cryosections of frozen tissue specimens were stained with hematoxylin-eosin

and evaluated for tumor cell content. Only the tumor samples that contained at least 50% of tumor cells on a microscopic section were used for further processing. Consequently, 151 pairs of cancerous and matched unaffected lung tissues were selected for the study. Clinicopathologic data and previously detected EGFR, KRAS, and HER2 gene mutational status were available for all the patients. For survival analysis, the overall survival (OS) was estimated as the time from the date of the surgery to the date of death due to lung cancer recurrence or metastases (event) or to the date of the last control visit (censoring). The disease-free survival (DFS) was defined as the time from the date of the surgery to the date of disease ZD1839 nmr relapse or death, whichever occurred first (events), or to the date of the last visit (censoring). The study was approved by the Ethics Committee of the University, and written informed consent for specimen collection was obtained from each patient before the surgery. DNA and RNA were isolated simultaneously using a magnetic extraction method. Briefly, about 40 to 50 mg of tissue was disrupted in lysis buffer (Biomerieux, Marcy l’Etoile, France) with TissueRupter

(Qiagen, Hilden, Germany) and incubated with Proteinase K for 2 hours at 56°C. Nucleic acids from deproteinated cell lysates were extracted automatically on the EasyMag machine 17-AAG in vitro (bioMérieux) according to the producer’s protocol. Both DNA and RNA were present in the 100-μl resulting extracts. Nucleic acid quality was assessed electrophoretically. For gene expression analysis, RNA was transcripted into cDNA in a reaction with High Capacity RNA-to-cDNA

Master Mix (Applied Biosystems, Foster City, CA) according to the producer’s recommendations. MET CN was analyzed by a quantitative real-time duplex CYTH4 polymerase chain reaction (qPCR) on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) with a commercially available predesigned MET TaqMan Copy Number Assay (Hs0143282_cn) and a Reference RNase P Assay (PN4412907), both from Applied Biosystems. The qPCR was done in a 20-μl reaction mixture containing 10 μl of Applied Biosystems TaqMan Universal PCR Master Mix with UNG, 1 μl of the CN assay solution, 1 μl of the reference assay solution, and 5 μl of DNA solution according to the following cyclic conditions: 50°C for 2 minutes followed by holding for 10 minutes at 95°C and 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. Each sample was analyzed in quadruplicate. The raw post-PCR data were used for MET CN calculation by the relative quantification method using the CopyCaller v.1.

305/N-C0ST/2008/0). The project

was carried out by a partnership of the Branch of Marine Geology of the Polish Geological Institute – National Research Institute, the Maritime Institute in Gdańsk and the Marine Fisheries Institute – National Research Institute, and in close cooperation with the Maritime Office in Gdynia. Investigations of meio- and macrozoobenthos were performed by the Marine Fisheries Institute – National Research Institute, and their results are to be presented in separate publications. PF-562271 molecular weight The authors would like to thank the reviewers for their valuable remarks and comments. The authors especially appreciate the efforts of reviewer Dr Adam Kubicki from Senckenberg am Meer, Wilhelmshaven, whose comments and suggestions significantly improved the paper. “
“The importance of polychaetes as feed in aquaculture is attributed to their potential to provide polyunsaturated fatty acids, which are essential for egg maturation in cultured prawns (Meunpol

et al. 2005, Nguyen et al. 2012), spawning in hatchery-reared fish species selleck screening library (Dinis et al. 1996) and enhancing reproductive performance in reared prawn stocks (Huang et al. 2008). Pseudonereis anomala Gravier 1901 is an Indo-Pacific nereid polychaete species that migrated through the Suez Canal from the Red Isotretinoin Sea into the Mediterranean and established healthy populations ( Çinar & Altun 2007, Dorgham et al. 2013). It can

act as a food source for many large predators, including crabs and fishes ( Çinar & Altun 2007), as it occurs in a variety of shallow water benthic habitats ( Ergen & Çinar 1997, Çinar & Ergen 2005) and exhibits a wide ecological valence that enables it to extend its distributional range into different parts of the Mediterranean ( Çinar & Altun 2007). Polychaetes are widely used as bait in recreational fishing in Egypt, but they are not applied as feed in aquaculture owing to the lack of information about their nutritional value. Such information is not available because little attention has been paid to the biochemical composition of polychaetes along Egyptian coasts. Only Osman (2007) measured protein and total lipids in the Oenonid polychaete Halla parthenopeia from the Suez Canal. The present study aims to measure the amount of some biochemical components in P. anomala in order to assess its potential as a source of fatty acids and amino acids for animal feeds in aquaculture. The worms were collected seasonally (summer: August, autumn: October, winter: January and spring: April) from hard substrates within a depth range of 20–50 cm on the Alexandria coast from August 2009 to July 2010.

The polymerization of the resin subsequently proceeded at 60 °C for 48 h. The embedded samples were sectioned BYL719 chemical structure in ∼70 nm thick slices using a diamond knife. The sections were transferred to supported gold grids and stained with uranyl acetate and Pb-citrate. The samples were observed under a Carl-Zeiss Model LEO906 transmission electron microscope. The surface-response methodology was used to study the effect of the plasticizer concentration (Cg or Cs) and process temperature (Tp) on dependent variables (mechanical properties and solubility).

The levels of the independent variables were defined according to a 22 full-factorial central composite design (star configuration) (Table 1 and Table 2).

An analysis of variance (ANOVA), a multiple comparison test, and all statistical analyses were performed using the Statistica 6.0 software. The data were fitted to a second order equation (Eq. (2)) as a function of the independent variables. equation(2) Yi=b0+b1X1+b2X2+b12X1X2+b11X11+b22X22where bn are constant regression coefficients, Yi are dependent variables (puncture force (PF), puncture deformation (PD), tensile strength (TS), elongation at break (E), Young’s modulus (YM), and solubility (S)), and X1 and X2 are the coded independent variables (plasticizer concentration and process temperature, respectively). After the surface-response results, were obtained, Avelestat (AZD9668) optimization

of the process conditions was carried out by multi-response analysis TSA HDAC (Derringer & Suich, 1980). This method involves the transformation of response variables (Yi) to an individual function of dimensionless desirability (gi) (Eq. (4)), ranging from 0 (undesirable response) to 1 (desired response). From the geometric means of individual desires, the overall desirability function (G) (Eq. (3)) is obtained. G was later maximized using the software Mathematic 5.0. equation(3) G=(g1n1,g2n2,……,gknk)1/kwhere: equation(4) gi=Yi−YminYmax−Yminwhere Ymin is the response minimum value and Ymax is the response maximum value, k is the number of considered responses, and ni is the weight of each response. In the case of solubility, Eq. (4) had to be redesigned, so that the minimum values for these responses could be obtained (Eq. (5)). equation(5) gi=Ymax−YiYmax−Ymin Finally, the Tukey’s test was applied at a 5% significance level to compare means for mechanical, solubility, moisture content, barrier properties, and GAB parameters of glycerol and sorbitol films prepared using the optimal formulation. The amaranth flour contains 9.0 ± 0.4 g/100 g moisture, 2.1 ± 0.0 g/100 g ash, 7.9 ± 0.2 g/100 g lipids, 14.1 ± 0.3 g/100 g protein, and 75.8 ± 0.2 g/100 g starch (among which 11.9 ± 0.3 g/100 g was amylose) (dry basis).

Venom was collected according to da Silveira et al. (2002), pooled and stored at −20 °C until use. Protein concentration was determined by Bradford method ( Bradford, 1976). L. laeta, Loxosceles intermedia and Loxosceles gaucho Brazilian mature spiders were collected in the region of Curitiba, PR, Brazil and maintained at the Centro de Produção e Pesquisa de Imunobiológicos (CPPI) of the State of Paraná, Brazil. The venoms from mature spiders were obtained as described before. Phoneutria nigriventer spiders and Tityus serrulatus scorpions were collected in the region of Belo

Horizonte and maintained at the “Seção de Animais Peçonhentos” of Ezequiel Dias Foundation (FUNED) of Belo Horizonte, Brazil. The crude

venoms were obtained by electric stimulation, lyophilized and stored at −20 °C in the dark until use. Two commercial antivenoms were used for the neutralization assay, the antivenom produced in CPPI, Brazil ABT-199 order (Lot. S02100) against BLlv, L. intermedia and L. gaucho venoms and an antivenom produced by Instituto Nacional de Salud del Perú (INS) (Lot. 0300069), containing antibodies against PLlv. The lethality was assessed via intradermal (i.d.) route. Groups of four mice were injected with different doses of venoms (0.4, 0.56, 0.784, 1.098, 1.537, 2.152 mg per kg of body weight) dissolved in 0.1 mL of PBS-BSA 0.5%. Seventy-two hours later deaths were recorded and the LD50 was then calculated by Probit analysis (Finney, 1971). The dermonecrotic, hemorrhagic selleck inhibitor and edematogenic activities of PLlv and BLlv were determined by intradermal injection of 10 μg of crude venom in 100 μL of PBS pH 7.2 into the shaved back of

five rabbits for each venom, as described by Furlanetto (1962). Injection of PBS alone was used as negative control. The diameters of dermonecrotic, hemorrhagic and edematogenic lesions were measured in the skin areas with a scale meter and caliper rule, 72 h after injection. Three measures of each lesion were made and their arithmetic mean was considered the mean diameter of the lesion. The sphingomyelinase (SMase) activity was measured using the Amplex Red Sphingomyelinase Assay Kit (Invitrogen) as previously described (Gatt et al., 1978; Binford et al., 2009). Briefly, different amounts isometheptene of the venoms (0.125, 0.25, 0.5, 1.0 μg) were assayed in triplicates. Varion Cary eclipse fluorescence spectrophotometer was used to measure the fluorescence emission from the reactions. Protein profile of PLlv and BLlv was analyzed by two-dimensional electrophoresis using the IPG-SDS-PAGE system ( Gorg, 1993). The venom was solubilized in lysis buffer containing 8 M urea, 2 M thiourea, 4% w/v CHAPS, 65 mM dithioerythritol, 40 mM Tris, 0.002% bromophenol blue, protease inhibitor and 1% of IPG buffer. Nonlinear immobilized pH 3–10 gradient IPG strips were rehydrated with 100 μg of the venom for 4 h (no electric field) and then for 12 h at 30 V.