| 1. |
- Adamczyk, Barbara, 1985, et al.
(författare)
-
High-throughput analysis of the plasma N-glycome by UHPLC
- 2017
-
Ingår i: High-Throughput Glycomics and Glycoproteomics, Eds: Gordan Lauc & Manfred Wuhrer. - New York : Springer. - 1064-3745. - 9781493964918 ; , s. 97-108
-
Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- The understanding of glycosylation alterations in health and disease has evolved significantly and glycans are considered to be relevant biomarker candidates. High-throughput analytical technologies capable of generating high-quality, large-scale glycoprofiling data are in high demand. Here, we describe an automated sample preparation workflow and analysis of N-linked glycans from plasma samples using hydrophilic interaction liquid chromatography with fluorescence detection on an ultrahigh-performance liquid chromatography (UHPLC) instrument. Samples are prepared in 96-well plates and the workflow features rapid glycoprotein denaturation, enzymatic glycan release, glycan purification on solid-supported hydrazide, fluorescent labeling, and post-labeling cleanup with solid-phase extraction. The development of a novel approach for plasma N-glycan analysis and its implementation on a robotic platform significantly reduces the time required for sample preparation and minimizes technical variation. It is anticipated that the developed method will contribute to expanding high-throughput capabilities to analyze protein glycosylation. © Springer Science+Business Media New York 2017.
|
|
| 2. |
- Ahlenius, Henrik
(författare)
-
Preface
- 2021
-
Ingår i: Methods in Molecular Biology. - 1064-3745. ; 2352
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
|
|
| 3. |
- Ahmad, Faiyaz, et al.
(författare)
-
Adenovirus-mediated overexpression of murine cyclic nucleotide phosphodiesterase 3B
- 2005
-
Ingår i: Methods in molecular biology (Clifton, N.J.). - New Jersey : Humana Press. - 1940-6029 .- 1064-3745. ; 307, s. 93-107
-
Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- To construct the recombinant adenovirus vector containing the cDNA for recombinant mouse cyclic nucleotide phosphodiesterase 3B (mPDE3B), the cDNA for mPDE3B was subcloned into pACCMV.pLpA. Subsequently, this recombinant plasmid, pACCMV.mPDE3B, was cotransfected with pJM17 plasmid containing the adenoviral genome into 293 human embryonic kidney cells, and the replication-deficient adenovirus AdCMV.mPDE3B was generated via homologous recombination. Large-scale preparation of adenovirus yielded 10(11)-10(13) viral particles/mL and could be quantitated by real-time polymerase chain reaction using iCycler (Bio-Rad). Efficiency of gene transfer was assessed by infecting FDCP2 or H4IIE cells with a recombinant adenovirus expressing beta-galactosidase (beta-gal); greater than 75% of cells were infected. Expression of mPDE3B in H4IIE hepatoma cells, FDCP2 hematopoietic cells, and beta-cells from isolated pancreatic islets was detected by Western blot analysis. In lysates from FDCP2 cells and H4IIE hepatoma cells infected with recombinant adenoviral mPDE3B constructs, mPDE3B activity was increased 10- to 30-fold compared with the activity in lysates from cells infected with beta-gal adenovirus. Stimulation of FDCP2 cells infected with mPDE3B adenovirus with insulin (100 nM, 10 min) resulted in an approx 1.7-fold increase in endogenous PDE3B and recombinant wild-type PDE3B activities. Infection of rat pancreatic islets resulted in a 5- to 10-fold increase in PDE3B expression and activity and subsequent blunting of insulin secretion. Thus, adenovirus-mediated gene transfer is effective for studying expression and regulation of recombinant PDE3 in insulin-responsive cells as well as insulin-secreting cells.
|
|
| 4. |
- Ahmed, RK, et al.
(författare)
-
T-cell epitope mapping
- 2009
-
Ingår i: Methods in molecular biology (Clifton, N.J.). - Totowa, NJ : Humana Press. - 1064-3745. ; 524, s. 427-38
-
Tidskriftsartikel (refereegranskat)
|
|
| 5. |
- Aits, Sonja, et al.
(författare)
-
Methods to Detect Loss of Lysosomal Membrane Integrity
- 2019
-
Ingår i: Autophagy : Methods and Protocols - Methods and Protocols. - New York, NY : Springer New York. - 1064-3745. - 9781493988730 ; 1880, s. 315-329
-
Bokkapitel (refereegranskat)abstract
- Loss of lysosomal membrane integrity, often referred to as lysosomal membrane permeabilization (LMP), occurs in many instances of cell death either as an initiating or as an amplifying event. Currently, the best method for detecting LMP is the galectin puncta formation assay which can be used for a broad range of sample types, both fixed and live, is easy to perform, and highly sensitive. This method, which is similar to the widely used LC3 puncta formation assay for autophagy, is based on the translocation of galectins to damaged lysosomes resulting in a change from uniform to punctate staining pattern. Here, we provide protocols for the galectin puncta formation assay in fixed and live cells and for an alternative assay based on fluorescent dextran release from damaged lysosomes, which can be performed in parallel
|
|
| 6. |
|
|
| 7. |
- Allen, Marie, et al.
(författare)
-
Mitochondrial D-loop and coding sequence analysis using pyrosequencing
- 2005
-
Ingår i: Methods in Molecular Biology. - New Jersey : Humana Press. - 1064-3745 .- 1940-6029. ; 297, s. 179-196
-
Tidskriftsartikel (refereegranskat)abstract
- In forensic casework analysis, mitochondrial deoxyribonucleic acid (DNA) often is used when the evidence material contains scarce amounts of DNA. Here, a mitochondrial DNA typing system for D-loop and coding region analysis based on pyrosequencing is described. Pyrosequencing is a real-time, single-tube sequencing-by-synthesis method, in which a cascade of enzymatic reactions yields detectable light. This pyrosequencing system has a higher resolution than the D-loop analysis performed routinely today as it also covers informative positions in the mitochondrial coding region. The system is composed of 16 polymerase chain reaction (PCR) fragments and 24 pyrosequencing reactions with a turn around time for a 96-well plate of less than 3 h after PCR.
|
|
| 8. |
- Allen, Marie, et al.
(författare)
-
Universal tag arrays in forensic SNP analysis.
- 2005
-
Ingår i: Methods in Molecular Biology. - 1064-3745 .- 1940-6029. ; 297, s. 141-154
-
Tidskriftsartikel (refereegranskat)abstract
- Microarray-based single nucleotide polymorphism (SNP) genotyping enables simultaneous and rapid detection of a large number of markers and is thus an attractive method for forensic individual acid identification. This assay relies on a one-color detection system and minisequencing in solution before hybridization to universal tag arrays. The minisequencing reaction is based on incorporation of a fluorescent dideoxynucleotide to a primer containing a tag-sequence flanking the position to be interrogated. This one-color system detects C and T polymorphisms in separate reactions on multiple polymerase chain reaction targets with the fluorophore TAMRA coupled to the respective dideoxynucleotide. After incorporation, tagged primer sequences are hybridized through their complementary sequence on the array, and positive signals are detected by a confocal laser-scanner.
|
|
| 9. |
- Altai, Mohamed, et al.
(författare)
-
Preparation of Conjugates for Affibody-Based PNA-Mediated Pretargeting.
- 2020
-
Ingår i: Methods in Molecular Biology. - New York, NY : Springer US. - 1064-3745 .- 1940-6029. ; 2105, s. 283-304, s. 283-304
-
Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are small engineered scaffold proteins suitable for in vivo tumor targeting. Radionuclide molecular imaging using directly radiolabelled affibody molecules provides excellent imaging. However, affibody molecules have a high renal reabsorption, which complicates their use for radionuclide therapy. The high renal reabsorption is a common problem for the use of engineered scaffold proteins for radionuclide therapy. Affibody-based PNA-mediated pretargeting reduces dramatically the absorbed dose to the kidneys and makes affibody-based radionuclide therapy possible. This methodology might, hopefully, solve the problem of high renal reabsorption for radionuclide therapy mediated by other engineered scaffold proteins.
|
|
| 10. |
- Alvarez-Castro, Jose, et al.
(författare)
-
Estimation and interpretation of genetic effects with epistasis using the NOIA model.
- 2012
-
Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1064-3745 .- 1940-6029. ; 871, s. 191-204
-
Tidskriftsartikel (refereegranskat)abstract
- We introduce this communication with a brief outline of the historical landmarks in genetic modeling, especially concerning epistasis. Then, we present methods for the use of genetic modeling in QTL analyses. In particular, we summarize the essential expressions of the natural and orthogonal interactions (NOIA) model of genetic effects. Our motivation for reviewing that theory here is twofold. First, this review presents a digest of the expressions for the application of the NOIA model, which are often mixed with intermediate and additional formulae in the original articles. Second, we make the required theory handy for the reader to relate the genetic concepts to the particular mathematical expressions underlying them. We illustrate those relations by providing graphical interpretations and a diagram summarizing the key features for applying genetic modeling with epistasis in comprehensive QTL analyses. Finally, we briefly review some examples of the application of NOIA to real data and the way it improves the interpretability of the results.
|
|
