The choice of an antibody for a protein-based research study is one of the most crucial steps in any project. Seemingly straightforward, the process is actually quite nuanced and filled with ...potential pitfalls. In this chapter, we will discuss five major topics that require consideration in the antibody selection process. These include overall study objectives and resources, details of both species and clonality, suitability in applications, and available detection methods. Each section will provide background information on the topic as well as specifics of antibody use in the laboratory. This chapter may be used as a guide to help vet antibody candidates for your project so you can stain with confidence.
•Edible insects are increasingly promoted as an efficient and sustainable food source.•Even if allergy to insects by ingestion is recognized, only a few studies are actually available.•Allergenicity ...of arginine kinase (AK) from mealworms and crickets is comparatively investigated.•Whole insects and purified proteins sources show different allergenic reactions based on entomological staff sera.•Low specific allergenicity to AK is discussed in relation to insect safety for human food.
Insects are seen as a solution to the increasing demand for protein sources for food. However, entomophagy has unfortunately been linked to allergic reactions in Europe with people with professional contacts. As mealworms (Tenebrio molitor) and crickets (Acheta domesticus) have recently become commercially available (both whole or in food formulation) in several European countries, this research assessed the cross allergenicity of arginine kinase (AK). Based on the collection of sera from a entomology laboratory staff, oven cooked insects but also purified AK fractions were tested. Immunoblotting against the protein extracts revealed different Immunoglobulin E reactivity of sera according to the insect target species: two bands (40 and 14 kDa) for crickets and a pattern including light responses at 17, 25 and 37 kDa for mealworms. Focusing on AK, low specific allergenicity was here illustrated and discussed in relation to the development of a safe edible insect consumption by humans.
Les sarcoglycanopathies sont des maladies musculaires génétiques à transmission autosomique récessive. Elles sont dues à une déficience d’une protéine du complexe sarcoglycane entraînant une ...instabilité de la membrane cellulaire.
Étudier les caractéristiques phénotypiques et immunologiques des différents types des sarcoglycnopathies par étude immunohistochimique (IHC) et par Western Blot Multiplex (WB).
Notre étude est menée sur 29 patients appartenant à 18 familles tunisiennes présentant les différents types des sarcoglycanopathies. Nous avons étudié les caractéristiques cliniques de nos patients. L’atteinte cardiaque et respiratoire était recherchée par la réalisation d’une échographie cardiaque et d’une épreuve fonctionnelle respiratoire. La déficience protéique en protéines du complexe sarcoglycane chez nos patients a été démontrée par IHC et par WB.
Par l’IHC et WB nous avons révélé l’absence des sarcoglycanes : α chez 9 patients, β chez 3 patients et la γ chez 11 patients. Aucune déficience n’a été détectée pour la sarcoglycane δ. Cependant, nous avons détecté l’absence de plus d’une protéine chez 6 patients. L’atteinte clinique était variable chez nos patients : 67 % des patients avaient une hypertrophie des mollets, une cardiomyopathie était notée chez 21 % patient et un seul patient avait une macroglossie.
Conformément aux données de la littérature, la sarcoglycanopathie γ était la forme la plus fréquente dans notre série. Une variabilité phénotypique était notée chez nos patients. L’atteinte cardiaque était aussi fréquente dans notre groupe. L’immunomarquage seul ne peut pas être spécifique d’un déficit protéique. Dans ce cas, une étude génétique s’avère indispensable pour la confirmation de diagnostic.
Malgré la similarité de phénotype entre les sarcoglycanopathies certaines particularités permettent de les distinguer. Une étude immunologique était indispensable pour orienter le diagnostic. L’étude génétique reste nécessaire pour le confirmer.
The objective of this study was to analyze the immune responses of bucks to small ruminant lentivirus (SRLV) with a focus on the reproductive system of males with recent and chronic infection. A ...total of 12 bucks were selected, six seronegative and six seropositive with chronic natural infection for more than 18 months (chronic infection group). After selecting the animals, the six seronegative males were intravenously inoculated with caprine arthritis-encephalitis virus (CAEV)-Co viral strain at a titer of 10-5,6 TCID50/mL. After viral inoculation, this group was called the recent infection group and was monitored weekly with the chronically infected group for 180 days with blood serum and seminal plasma Western Blot (WB) analysis. Of the animals with chronic SRLV infection, 18.94% (50/264) showed anti-SRLV antibodies in at least one of the samples, and 81.06% (214/264) were negative. Anti-SRLV antibodies were detected in 27.27% (36/132) of the blood serum samples from this group, while 10.60% (14/132) were reactive in the seminal plasma WB test. The animals inoculated with CAEV-Co became seropositive after the third week of viral inoculation. In this group, 31.06% (41/132) of seminal plasma samples had anti-SRLV antibodies, and of these, 70.73% (29/41) coincided with blood serum results. Of the remaining 29.27% (12/41), the seminal plasma sample of only three animals (RIA2, RIA3, and RIA5) had anti-SRLV antibodies. One of the animals with a recent infection presented anti-SRLV antibodies only in seminal plasma samples, possibly due to virus compartmentalization. Intermittent viral shedding was observed in both biological samples, regardless of the infection stage. The immune response in bucks with recent SRLV infection is more significant than in chronically infected animals. Regardless of the stage of infection, there is a fluctuation in antibody levels, therefore, this creates a risk of false-negative samples when performing the diagnosis.
Since its first description, Western blot has been widely used in molecular labs. It constitutes a multistep method that allows the detection and/or quantification of proteins from simple to complex ...protein mixtures. Western blot quantification method constitutes a critical step in order to obtain accurate and reproducible results. Due to the technical knowledge required for densitometry analysis together with the resources availability, standard office scanners are often used for the imaging acquisition of developed Western blot films. Furthermore, the use of semi-quantitative software as ImageJ (Java-based image-processing and analysis software) is clearly increasing in different scientific fields. In this work, we describe the use of office scanner coupled with the ImageJ software together with a new image background subtraction method for accurate Western blot quantification. The proposed method represents an affordable, accurate and reproducible approximation that could be used in the presence of limited resources availability.
In this new era of precision medicine, characterization of single-cell subpopulations to better understand disease etiology is paramount. It is thus an opportune time to explore techniques that allow ...molecular analysis of single cells and to better understand the basis of pathogenesis of diseases like cancer. Single-cell western blotting is one such method that allows analysis of single cells at the protein level. In contrast to traditional western blotting, which relies heavily on bulk analysis of lysates generated from tissues and is often indicative of the population average, this technique allows analysis of lysates from single-cell subpopulations thereby providing a glimpse into cell heterogeneity. The method entails the use of a chip containing 30 μm thick photoactivated polyacrylamide gel spotted with nearly 6400 microwells. Single cells loaded on the chip are captured in the microwells by passive gravity and are then lysed and electrophoresed using the MILO™ single-cell western platform. This method forgoes the use of transfer of proteins on a PVDF and a nitrocellulose membrane, as performed in traditional western blotting, and all other steps including probing of primary and fluorescent secondary antibodies against the protein of interest are performed directly on the chip. The proteins of interest can then be visualized by scanning a chip with the use of a microarray scanner. The entire procedure can be performed in as less as 4-6 h, and thus this method provides several advantages over traditional western blotting.
The hypoxia-inducible transcription factors HIF-1 and HIF-2 regulate the response to hypoxia. Both proteins are dimers of an alpha subunit and a shared beta subunit. Under hypoxic conditions, the ...alpha subunits are stabilized, and the transactivation ability of the HIF-1 transcription factor is induced. Accordingly, assessment of HIF-1α protein levels and HIF transcriptional activity serve as an indirect indicator of hypoxia. In this series of protocols, I describe three methods to probe the HIF pathway.
Sturgeons are highly valuable species from both a conservation and economic point of view. Their very sought-after caviar extracted from mature females has both a rich culture around it and opens the ...way for interesting research. Because of the advanced age at which females and males could be identified in aquaculture it is important to develop a set of molecular markers that could make the distinction easy. For this, a better understanding of the sexual development of sturgeons is necessary. We selected the Dmrt1 (doublesex and mab-3 related transcription factor 1) and the Cyp17a1 (17α-hydroxylase) markers with the scope of investigating the gonad development of sturgeon individuals. Both markers appear to be expressed in the gonad but no statistically significant difference in expression is observed between females and males. This could mean that the markers are equally involved in female and male sexual development at the stage of sampling. This study paves the way for a better understanding of sturgeon sexual development.
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