Immuno-based Detection Assays to Quantify Distinct Mutant Huntingtin Conformations in Biological Samples

2013 ◽  
pp. 163-171 ◽  
Author(s):  
Gregor P. Lotz ◽  
Andreas Weiss
Keyword(s):  
Biological Samples ◽  
Mutant Huntingtin

Reliable Resolution of Full-Length Huntingtin Alleles by Quantitative Immunoblotting

2021 ◽  
pp. 1-11
Author(s):  
Nicholas S. Caron ◽  
Christine Anderson ◽  
Hailey Findlay Black ◽  
Shaun S. Sanders ◽  
Fanny L. Lemarié ◽  
...  
Keyword(s):  
Huntington's Disease ◽  
Biological Samples ◽  
Antisense Oligonucleotide ◽  
Clinical Development ◽  
Full Length ◽  
Preclinical Studies ◽  
Mutant Huntingtin ◽  
Wild Type ◽  
Wide Range ◽  
Quantitative Immunoblotting

Background: Therapeutics that lower mutant huntingtin (mHTT) have shown promise in preclinical studies and are in clinical development for the treatment of Huntington’s disease (HD). Multiple assays have been developed that either quantify mHTT or total HTT but may not accurately measure levels of wild type HTT (wtHTT) in biological samples. Objective: To optimize a method that can be used to resolve, quantify and directly compare levels of full length wtHTT and mHTT in HD samples. Methods: We provide a detailed quantitative immunoblotting protocol to reproducibly resolve full length wtHTT and mHTT in multiple HD mouse and patient samples. Results: We show that this assay can be modified, depending on the sample, to resolve wtHTT and mHTT with a wide range of polyglutamine differences (ΔQs 22–179). We also demonstrate that this method can be used to quantify allele-selective lowering of mHTT using an antisense oligonucleotide in HD patient-derived cells. Conclusion: This quantitative immunoblotting method can be used to reliably resolve full length HTT alleles with ΔQs≥22 and allows for direct comparison of wtHTT and mHTT levels in HD samples.

An Efficient Statistic for Determining the Diameter of Thin Sectioned Uniform Spheres from Measurements of Biological Samples

1974 ◽  
Vol 32 ◽  
pp. 114-115
Author(s):  
W. R. Schucany ◽  
G. H. Kelsoe ◽  
V. F. Allison
Keyword(s):  
Biological Samples ◽  
Traditional Method ◽  
Cell Types ◽  
Accurate Estimation ◽  
Morphological Identification ◽  
Sectioned Material ◽  
Maximal Diameter ◽  
Similar Cell

Accurate estimation of the size of spheroid organelles from thin sectioned material is often necessary, as uniquely homogenous populations of organelles such as vessicles, granules, or nuclei often are critically important in the morphological identification of similar cell types. However, the difficulty in obtaining accurate diameter measurements of thin sectioned organelles is well known. This difficulty is due to the extreme tenuity of the sectioned material as compared to the size of the intact organelle. In populations where low variance is suspected the traditional method of diameter estimation has been to measure literally hundreds of profiles and to describe the “largest” as representative of the “approximate maximal diameter”.

The Application of Ultra-Thin Sectioning Techniques to Non-Biological Samples

1968 ◽  
Vol 26 ◽  
pp. 332-333
Author(s):  
C. F. Oster
Keyword(s):  
Biological Sciences ◽  
Epoxy Resin ◽  
Cross Sections ◽  
Biological Samples ◽  
Industrial Applications ◽  
Photographic Film ◽  
Silver Particles ◽  
Thin Sectioning ◽  
Embedding Medium

Although ultra-thin sectioning techniques are widely used in the biological sciences, their applications are somewhat less popular but very useful in industrial applications. This presentation will review several specific applications where ultra-thin sectioning techniques have proven invaluable.The preparation of samples for sectioning usually involves embedding in an epoxy resin. Araldite 6005 Resin and Hardener are mixed so that the hardness of the embedding medium matches that of the sample to reduce any distortion of the sample during the sectioning process. No dehydration series are needed to prepare our usual samples for embedding, but some types require hardening and staining steps. The embedded samples are sectioned with either a prototype of a Porter-Blum Microtome or an LKB Ultrotome III. Both instruments are equipped with diamond knives.In the study of photographic film, the distribution of the developed silver particles through the layer is important to the image tone and/or scattering power. Also, the morphology of the developed silver is an important factor, and cross sections will show this structure.

Artefacts in the x-ray microanalysis of frozen-hydrated biological samples

1987 ◽  
Vol 45 ◽  
pp. 658-659
Author(s):  
Patrick Echlin
Keyword(s):  
Electron Scattering ◽  
Biological Samples ◽  
Fracture Face ◽  
Detailed Structure ◽  
X Ray ◽  
Morphological Appearance ◽  
The Poor ◽  
Freeze Dried

A number of papers have appeared recently which purport to have carried out x-ray microanalysis on fully frozen hydrated samples. It is important to establish reliable criteria to be certain that a sample is in a fully hydrated state. The morphological appearance of the sample is an obvious parameter because fully hydrated samples lack the detailed structure seen in their freeze dried counterparts. The electron scattering by ice within a frozen-hydrated section and from the surface of a frozen-hydrated fracture face obscures cellular detail. (Fig. 1G and 1H.) However, the morphological appearance alone can be quite deceptive for as Figures 1E and 1F show, parts of frozen-dried samples may also have the poor morphology normally associated with fully hydrated samples. It is only when one examines the x-ray spectra that an assurance can be given that the sample is fully hydrated.

Utilization of High Performance Ion Chromatography (HPIC) method for the determination of total sulphur content in different biological samples

Planta Medica ◽  
2011 ◽  
Vol 77 (12) ◽  
Author(s):  
A Sapcanin ◽  
E Kovac Besovic ◽  
M Pazalja ◽  
D Kresic ◽  
E Mujic ◽  
...  
Keyword(s):  
Ion Chromatography ◽  
Biological Samples ◽  
High Performance ◽  
Sulphur Content ◽  
Total Sulphur ◽  
Total Sulphur Content

A Fast and Sensitive Method Combining Reversed-Phase Chromatography with High Resolution Mass Spectrometry to Quantify 2-Fluoro-2-Deoxyglucose and Its Phosphorylated Metabolite for Determining Glucose Uptake

2019 ◽  
Author(s):  
Ashley Williams ◽  
Deborah Muoio ◽  
Guofang Zhang
Keyword(s):  
Glucose Uptake ◽  
Biological Samples ◽  
High Resolution Mass Spectrometry ◽  
Reversed Phase ◽  
Sodium Adduct ◽  
Negative Mode ◽  
Glucose Analogues ◽  
Positive Mode ◽  
Q Exactive ◽  
Glucose Analogue

Quantative measurements of the glucose analogue, 2-deoxyglucose (2DG), and its phosphorylated metabolite (2-deoxyglucose-6-phosphate (2DG-6-P)) are critical for the measurement of glucose uptake. While the field has long identified the need for sensitive and reliable assays that deploy non-radiolabled glucose analogues to assess glucose uptake, no analytical MS-based methods exist to detect trace amounts in complex biological samples. In the present work, we show that 2DG is poorly suited for MS-based methods due to interfering metabolites. We therefore developed and validated an alternative C18-based LC-Q-Exactive-Orbitrap-MS method using 2-fluoro-2-deoxyglucose (2FDG) to quantify both 2FDG and 2FDG-6-P by measuring the sodium adduct of 2FDG in the positive mode and deprotonation of 2FDG-6-P in the negative mode. The low detection limit of this method can reach 81.4 and 48.8 fmol for both 2FDG and 2FDG-6-P, respectively. The newly developed method was fully validated via calibration curves in the presence and absence of biological matrix. The present work is the first successful LC-MS method that can quantify trace amounts of a nonradiolabeled glucose analogue and its phosphorylated metabolite and is a promising analytical method to determine glucose uptake in biological samples.

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