scholarly journals Optimization of Chemoenzymatic Mass Tagging by Strain-Promoted Cycloaddition (SPAAC) for the Determination of O-GlcNAc Stoichiometry by Western Blotting

Biochemistry ◽  
2018 ◽  
Vol 57 (40) ◽  
pp. 5769-5774 ◽  
Author(s):  
Narek Darabedian ◽  
John W. Thompson ◽  
Kelly N. Chuh ◽  
Linda C. Hsieh-Wilson ◽  
Matthew R. Pratt
Keyword(s):  
Western Blotting

scholarly journals Effect of anti-thyroid peroxidase (TPO) antibodies on TPO activity measured by chemiluminescence assay

1997 ◽  
Vol 43 (8) ◽  
pp. 1392-1396 ◽  
Author(s):  
V Kaczur ◽  
Gy Vereb ◽  
I Molnár ◽  
G Krajczár ◽  
E Kiss ◽  
...  
Keyword(s):  
Western Blotting ◽  
Competitive Inhibition ◽  
High Sensitivity ◽  
Inhibitory Effect ◽  
Thyroid Peroxidase ◽  
Chemiluminescence Detection ◽  
Chemiluminescence Method ◽  
Blotting Technique ◽  
Hypothyroid Patients

Abstract A chemiluminescence method was developed to measure thyroid peroxidase (TPO) activity and the inhibitory effect of anti-TPO antibodies in purified porcine TPO. The TPO preparation was characterized kinetically and controlled by Western-blotting technique. The chemiluminescence method proved to be reproducible and much more sensitive than the widely used guaiacol method, being able to detect TPO concentrations of 2.21 × 10−5 g/L vs 6.63 × 10−2g/L with the latter. Otherwise, the determinations with the two methods correlated well (r = 0.76). Investigating the effect of IgGs from 23 hypothyroid patients on measured TPO activity, we detected inhibition in 19 cases with the chemiluminescence technique (15 with the guaiacol method). Anti-TPO antibodies showed competitive inhibition of TPO activity with respect to the substrate guaiacol. In both systems, the inhibition is present in the IgG F(ab′)2 fragment. We conclude that the high sensitivity of chemiluminescence detection allows routine determination of the inhibition of TPO activity by anti-TPO antibodies.

scholarly journals The determination of immune reactive proteins inCysticercus tenuicolliscyst fluids by SDS-PAGE and Western blotting in sheep

Parasite ◽  
2003 ◽  
Vol 10 (2) ◽  
pp. 141-145 ◽  
Author(s):  
M. Kara ◽  
H.O. Sarimehmetoglu ◽  
B. Gonenc
Keyword(s):  
Western Blotting ◽  
Sds Page

Determination of Antibody Specificity by Western Blotting

Cell Biology ◽  
2006 ◽  
pp. 527-532 ◽  
Author(s):  
J CELIS ◽  
J MOREIRA ◽  
P GROMOV
Keyword(s):  
Western Blotting ◽  
Antibody Specificity

Determination of Copy Number of c-Myc Protein per Cell by Quantitative Western Blotting

1999 ◽  
Vol 269 (1) ◽  
pp. 66-71 ◽  
Author(s):  
Claudia Rudolph ◽  
Gerold Adam ◽  
Andreas Simm
Keyword(s):  
Western Blotting ◽  
Copy Number ◽  
Quantitative Western Blotting

Determination of monoclonal antibody specificity by immunoadsorption and Western blotting

1992 ◽  
Vol 152 (1) ◽  
pp. 59-67 ◽  
Author(s):  
Rolf Schuh ◽  
Elisabeth Kremmer ◽  
Evelyn Ego ◽  
Michael Wasiliu ◽  
Stefan Thierfelder
Keyword(s):  
Monoclonal Antibody ◽  
Western Blotting ◽  
Antibody Specificity

scholarly journals A simple secretion assay for assessing new and existing myocilin variants

2021 ◽  
Author(s):  
John Hulleman ◽  
Emi Nakahara
Keyword(s):  
Western Blotting ◽  
Primary Open Angle Glaucoma ◽  
Cultured Cells ◽  
Open Angle Glaucoma ◽  
Luciferase Assay ◽  
Sufficient Information ◽  
Aggregate Formation ◽  
Gaussia Luciferase ◽  
Secretion Assay

With the increasing use of molecular genetics approaches for determination of potential disease-causing mutations, it is becoming more important to be able to interpret and act upon the provided results. As an example of such an instance, nearly 300 mutations have been identified in the myocilin (MYOC) gene, which is the most commonly mutated gene causing primary open angle glaucoma. Yet a lack of sufficient information exists for many of these variants, hindering their definitive classification. While the function of MYOC is unclear, biochemically, the vast majority of glaucoma-causing MYOC mutations result in protein non-secretion and intracellular insoluble aggregate formation in cultured cells. Previously we generated a Gaussia luciferase-based MYOC fusion protein to sensitively track secretion of the protein. Herein we applied this same assay to fourteen clinically-derived MYOC variants with varying degrees of predicted pathogenicity and compared the luciferase secretion results with the better established MYOC assay of western blotting. Eight of the variants (G12R, V53A, T204T, P254L, T325T, D380H, D395_E396insDP, and P481S) had not been biochemically assessed previously. Of these, P254L and D395_E396insDP demonstrated significant secretion defects from human embryonic kidney (HEK-293A) cells reminiscent of glaucoma-causing mutations. Overall, we found that the luciferase assay results agreed with western blotting for thirteen of the fourteen variants (93%), suggesting a strong concordance. These results suggest that the Gaussia luciferase assay may be used as a complementary or standalone assay for quickly assessing MYOC variant behavior, and anticipate that these results will be useful in MYOC variant curation and reclassification.

scholarly journals A Novel Enzyme Capture - ELISA for the Direct Determination of Factor XIII Activity

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1174-1174
Author(s):  
Michael Andrew Durda ◽  
Amanda P. Waller ◽  
Kyle A Davis ◽  
Bryce A. Kerlin
Keyword(s):  
Western Blotting ◽  
High Throughput ◽  
Factor Xiii ◽  
Conflicts Of Interest ◽  
Direct Determination ◽  
Elisa Method ◽  
Transglutaminase Activity ◽  
Capture Elisa ◽  
Fxiii Activity

Abstract Background: Activated factor XIII (fXIII) mechanically stabilizes fibrin clots by catalyzing the formation of intramolecular crosslinks and retards plasmin-mediated fibrinolysis by incorporating intermolecular α2-antiplasmin (α2AP):fibrin crosslinks. High quality quantitative fXIII activity assays are required for the accurate and timely diagnosis of fXIII deficiency. Currently available high-throughput assays indirectly measure the transglutaminase activity of fXIII via detection of ammonia that is elaborated upon formation of ε-N-(γ-glutamyl)-lysyl crosslinking bonds. Unfortunately, these assays can be confounded by fXIII-independent ammonia producing and consuming reactions present in plasma. We thus set out to develop an assay which directly quantifies the transglutaminase activity. We hypothesized that fXIII could be captured in a microtiter plate with anti-fibrinogen antibodies and, following an activation step, its activity determined in a kinetic fashion via the rate of α2AP incorporation (measured in a subsequent ELISA step). Methods: Co-immunoprecipitation and western blotting techniques were utilized to confirm fXIII/fibrinogen interactions and to determine the presence of basal interactions between fibrinogen and α2AP. Simultaneously, this approach enabled the determination of suitable antibody pairs for use in the enzyme capture-ELISA (EC-ELISA). In microtiter plates, fibrinogen-captured fXIII was activated with excess thrombin, physiologic calcium, and GPRP peptide (to block fibrin polymerization) in the presence of excess α2AP. FXIII activity was stopped with a potent fXIII-inhibitor (iodoacetamide) at predetermined time points. Subsequently, α2AP incorporation was determined with a primary anti-α2AP antibody and secondary HRP-conjugated antibody. Results: Co-immunoprecipitation and western blotting experiments confirmed that both fXIII and α2AP circulate in complex with fibrinogen, as previously reported by others. However, α2AP incorporation significantly increased following incubation with activated factor XIII. Using the EC-ELISA method, we were able to demonstrate that α2AP is incorporated in a manner consistent with first order kinetics. Discussion: These experiments demonstrate the feasibility of an EC-ELISA assay to directly measure fXIII activity. Further work will be required to determine the sensitivity of this assay in test plasmas with varying known quantities of fXIII and how its performance characteristics compare to currently available indirect activity assays. With further optimization, the EC-ELISA method may lead to an improved, high throughput diagnostic assay for clinical use. Disclosures No relevant conflicts of interest to declare.

Galactic orbits of stars

1966 ◽  
Vol 25 ◽  
pp. 93-97
Author(s):  
Richard Woolley
Keyword(s):  
Globular Cluster ◽  
Potential Field ◽  
High Velocity ◽  
Velocity Vector ◽  
Variable Stars ◽  
The Sun ◽  
Proper Motions ◽  
Rr Lyrae ◽  
The Galaxy

It is now possible to determine proper motions of high-velocity objects in such a way as to obtain with some accuracy the velocity vector relevant to the Sun. If a potential field of the Galaxy is assumed, one can compute an actual orbit. A determination of the velocity of the globular clusterωCentauri has recently been completed at Greenwich, and it is found that the orbit is strongly retrograde in the Galaxy. Similar calculations may be made, though with less certainty, in the case of RR Lyrae variable stars.

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