Isolation of Pure Mitochondria from Rat Kidneys and WesternBlot of Mitochondrial Respiratory Chain Complexes
Cardiac, neuronal and renal tubular epithelial cells are the most metabolically active cells in the body. Their fate depends largely on their mitochondria as the primary energy generating system which participates in the control of apoptosis, cell cycle and metabolism. Thus, mitochondrial dysfunction is a hallmark of many chronic diseases including diabetic nephropathy. A drop in mitochondrial bioenergetics efficiency is often associated with altered expression of respiratory chain complexes. Moreover, recent studies demonstrate that cellular proteins can shuttle to mitochondria and modify their function directly.
Here we illustrate two mitochondria isolation protocols; one is recommended if the purity of the mitochondrial fraction is a priority such as if the mitochondrial localization of a protein has to be validated, the other if a high yield of intact functional mitochondria is required for functional studies and quantitative Western blotting. Next, we provide a detailed protocol for Western blotting of isolated mitochondria and renal cortex either to prove the purity of isolated fractions or to quantify complexes of the mitochondrial respiratory chain. We used this approach to identify classically cell membrane bound angiotensin II receptors in mitochondria and to study the effect of these receptors on mitochondrial function in early stages of diabetic nephropathy.
Identification of early diagnostic antigens in soluble proteins of Trichinella spiralis adult worms by Westernblot
Previous studies showed that crude antigens from Trichinella spiralis adult worms (AW) can be recognized by mouse infection sera at 8 days post infection. The aim of this study was to identify the early diagnostic antigenic bands in soluble proteins from T. spiralis AW by Western blot using early infection sera. The affecting factors of adult recovery were firstly observed in this study, and the results showed that the maximum number of adults was collected from small intestine when the female BALB/c mice were orally infected with 4000 ML and sacrificed at 3 days post infection.
The results of Western blot analysis showed that seven protein bands (31, 35.1, 39, 40.6, 41.9, 47 and 50.6 kDa) could be recognized by early infection sera as early as at 8-10 days post infection, and were strongly reacted with mouse infection sera at 11-12 days post infection. Our results suggested that the seven protein bands of T. spiralis AW soluble proteins might be the early expressed antigens during the intestinal stage of Trichinella infection and therefore have potential value for the early diagnosis of trichinellosis.
Tissue Inhibitor of Metalloproteinases 1, Recombinant, Mouse, Western Blot Control (TIMP1, Erythroid Potentiating Activity, EPA, Collagenase Inhibitor, CI)
Tissue Inhibitor of Metalloproteinases 1, Recombinant, Mouse, Western Blot Control (TIMP1, Erythroid Potentiating Activity, EPA, Collagenase Inhibitor, CI)
Simple Western: Bringing the WesternBlot into the Twenty-First Century
The Western blot is widely used in the study of protein biochemistry, but it is notoriously labor-intensive, and it is limited in its reproducibility and quantification, among many other challenges. By contrast, capillary-based protein separation and immunodetection, known as Simple Western™, overcomes many of the challenges associated with the traditional Western blot, and it is quickly gaining traction as a replacement for traditional Western blot analysis.
The advantages that capillary-based immunoassay offers include ease of use, automation, reproducibility, quantification, and even built-in total protein normalization. In this chapter, we describe protocols for the two basic types of capillary-based immunodetection assays, one by molecular weight separation and the other by charge separation. In both methods, protein samples are separated in the capillary followed seamlessly by immunodetection with chemiluminescent or fluorescent antibodies for highly sensitive and specific detection of target proteins.
Biological Validation of a Novel Process and Product for Quantitating WesternBlots
Protein normalization of western blots has relied upon housekeeping proteins which exhibit signal saturation and varied cellular expression level variations. These issues can produce spurious results leading to erroneous conclusions. A superior method to protein normalization using housekeeping proteins is Total Protein Normalization, a method now recognized as the gold standard for quantitative westerns.
Total Protein Normalization requires that all proteins on a membrane be stained or labeled uniformly, imaged, and then analyzed for total protein. It is important that such a normalization process not interfere with typical immunodetection methods, fits within existing western workflows, and exhibits a linear relationship of signal intensity to protein load under all experimental conditions. Here we report that we developed a new reagent enabling Total Protein Normalization, and we demonstrate its superior protein normalization capabilities through analysis of target proteins in different cell backgrounds.
These data illustrate how housekeeping proteins exhibit signal saturation, yield erroneous normalization data, and display sample-to-sample variations averaging 48.2% overall. Signal intensities obtained using our new method show a linear relationship to protein sample load, thus providing accurate protein normalization with an overall average variation of 7.7%.
Identification of Protein Carbonyls (PCOs) in Canine Serum by WesternBlot Technique and Preliminary Evaluation of PCO Concentration in Dogs With Systemic Inflammation
In people, serum Protein Carbonyls (PCOs) increase during oxidative stress (OS) due to oxidative damage to proteins. OS is often associated with inflammation and especially with sepsis, a condition hard to diagnose in veterinary medicine because reliable markers are lacking. The aim of this study was to assess whether PCOs in canine serum may be detected by antibody-based methods such as Western Blotting (WB), and to preliminarily investigate the possible utility of this marker in dogs with inflammation.
A serum sample oxidized in vitro was used to set up the method; the coefficient of variation obtained by repeated analysis varied from 24 to 36%. In order to assess whether the technique may cover the range of PCOs concentration detectable in routine practice, PCOs were measured in 4 healthy dogs and in 15 with inflammatory diseases, in some cases potentially associated with sepsis, as suggested by the results of other inflammatory markers such as C-Reactive Protein (CRP) and the anti-oxidant enzyme Paraoxonase 1 (PON-1): the concentration of PCOs was low in dogs with normal PON-1 activity,
moderately increased in the majority of dogs with low-normal PON-1 activity, and severely increased in dogs with very low PON-1 activity. In conclusion this study demonstrates that PCOs, may be detected in canine serum, using antibody-based techniques such as WB. The preliminary results in dogs with and without systemic inflammation encourage further studies on the possible role of PCOs as inflammatory markers.
