scholarly journals A Rapid and Facile Purification Method for Glycan‐Binding Proteins and Glycoproteins

2020 ◽  
Vol 101 (1) ◽  
Author(s):  
Christina J. Welch ◽  
Priyanka D. Kadav ◽  
Jared L. Edwards ◽  
Jessica Krycia ◽  
Melanie L. Talaga ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Purification Method

scholarly journals SPACE exploration of chromatin proteome to reveal associated RNA-binding proteins

2020 ◽  
Author(s):  
Mahmoud-Reza Rafiee ◽  
Julian A Zagalak ◽  
Giulia Tyzack ◽  
Rickie Patani ◽  
Jernej Ule ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Genome Maintenance ◽  
Purification Method ◽  
Comprehensive Knowledge ◽  
Associated Proteins ◽  
Versatile Technique ◽  
Dna Binders ◽  
Lateral Sclerosis

AbstractChromatin is composed of many proteins that mediate intermolecular transactions with the genome. Comprehensive knowledge of these components and their interactions is necessary for insights into gene regulation and other activities; however, reliable identification of chromatin-associated proteins remains technically challenging. Here, we present SPACE (Silica Particle Assisted Chromatin Enrichment), a stringent and straightforward chromatin-purification method that helps identify direct DNA-binders separately from chromatin-associated proteins. We demonstrate SPACE’s unique strengths in three experimental set-ups: the sensitivity to detect novel chromatin-associated proteins, the quantitative nature to measure dynamic protein use across distinct cellular conditions, and the ability to handle 10-25 times less starting material than competing methods. In doing so, we reveal an unforeseen scale of association between over 500 nuclear RNA-binding proteins (RBPs) with chromatin and DNA, providing new insights into their roles as important regulators of genome maintenance and chromatin composition. Applied to iPSC-derived neural precursors, we discover a new role for the amyotrophic lateral sclerosis (ALS)-causing Valosin Containing Protein (VCP) in recruiting DNA-damage components to chromatin, thus paving the way for molecular mechanistic insights into the disease. SPACE is a fast and versatile technique with many applications.

Purification of four gelatin-binding proteins from bovine seminal plasma by affinity chromatography

1987 ◽  
Vol 7 (3) ◽  
pp. 231-238 ◽  
Author(s):  
P. Manjunath ◽  
M. R. Sairam ◽  
J. Uma
Keyword(s):  
Seminal Plasma ◽  
Binding Proteins ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Apparent Molecular Weight ◽  
Single Step ◽  
High Yield ◽  
Property A ◽  
Purification Method ◽  
Elution Pattern

Bovine seminal plasma contains three similar acidic proteins, which we have previously designated as BSP-A1, BSP-A2, and BSP-A3. These proteins contain two homologous domains that are similar to type II structures present in the gelatin-binding domain of fibronectin. The present data have revealed that these proteins, like fibronectin, also form complexes with gelatin, a denatured collagen. Based on this property, a single step affinity purification method has been developed. In addition to these three proteins BSP-A1, −A2 and −A3, another protein with an apparent molecular weight of 30,000 dalton (named BSP-30-kDa) also bound to the gelatin-agarose column. Elution of these proteins from affinity columns using a linear gradient of either urea or arginine gave essentially the same pattern with a high yield of 90–95%. The purified proteins were homogeneous by SDS-polyacrylamide gel electrophoresis, amino acid composition and HPLC. Chromatography of bull seminal vesicular fluid also exhibited an elution pattern similar to that obtained for bull seminal plasma. The availability of these purified proteins should aid in understanding the physiology of these gelatin-binding proteins.

scholarly journals Effect of Multiple Mutations in the Hemoglobin- and Hemoglobin-Haptoglobin-Binding Proteins, HgpA, HgpB, and HgpC, ofHaemophilus influenzae Type b

1999 ◽  
Vol 67 (6) ◽  
pp. 2729-2739 ◽  
Author(s):  
Daniel J. Morton ◽  
Paul W. Whitby ◽  
Hongfan Jin ◽  
Zhen Ren ◽  
Terrence L. Stull
Keyword(s):  
Haemophilus Influenzae ◽  
Insertional Mutagenesis ◽  
Binding Proteins ◽  
Affinity Purification ◽  
Triple Mutant ◽  
Gene Encoding ◽  
Purification Method ◽  
Insertional Mutation ◽  
Ofhaemophilus Influenzae ◽  
Complete Deletion

ABSTRACT Haemophilus influenzae requires heme for growth and can utilize hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified two hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA and HgpB, in H. influenzae HI689. Insertional mutation of hgpA andhgpB, either singly or together, did not abrogate the ability to utilize or bind either hemoglobin or the hemoglobin-haptoglobin complex. A hemoglobin affinity purification method was used to isolate a protein of approximately 120 kDa from thehgpA hgpB double mutant. We have cloned and sequenced the gene encoding this third hemoglobin/hemoglobin-haptoglobin binding protein and designate it hgpC. Insertional mutation ofhgpC did not affect the ability of the strain to utilize either hemoglobin or hemoglobin-haptoglobin. An hgpA hgpB hgpC triple mutant constructed by insertional mutagenesis showed a reduced ability to use the hemoglobin-haptoglobin complex but was unaltered in the ability to use hemoglobin. A second class of mutants was constructed in which the entire structural gene of each of the three proteins was deleted. The hgpA hgpB hgpCcomplete-deletion triple mutant was unable to utilize the hemoglobin-haptoglobin complex and showed a reduced ability to use hemoglobin. We have identified three hemoglobin/hemoglobin-haptoglobin-binding proteins in Haemophilus influenzae. Any one of the three proteins is sufficient to support growth with hemoglobin-haptoglobin as the heme source, and expression of at least one of the three is essential for hemoglobin-haptoglobin utilization. Although the three proteins play a role in hemoglobin utilization, an additional hemoglobin acquisition mechanism(s) exists.

Uultrastructure of Microtubules

1972 ◽  
Vol 30 ◽  
pp. 252-253
Author(s):  
Ariaki Nagayama
Keyword(s):  
Fine Structure ◽  
Cultured Cells ◽  
Present Report ◽  
Confluent Monolayer ◽  
Purification Method ◽  
Vinca Rosea ◽  
L Cells ◽  
Antimitotic Activity ◽  
Monolayer Cultures ◽  
Rapid Purification

Vinblastine(Vb) or vincristine, alkaloid derived from Vinca rosea is known for its antimitotic activity by regrouping of microtubules into paracrystalline form within the cells. A rapid purification method of vinblastine-induced microtubular paracrystals(PC) has provided us with a fresh and pure microtubular material demonstrating the presence of a labile ATPase associated with the PC. The present report is concerned with the fine structure of purified microtubules of mammalian cultured cells.Confluent monolayer cultures of L cells were incubated for 20hrs with 10-5 M Vb (donated from Shionogi Seiyaku & Co., Osaka, Japan).

High resolution spot scan imaging of frozen, hydrated actin bundles with 400kV electrons

1992 ◽  
Vol 50 (1) ◽  
pp. 512-513
Author(s):  
J. Jakana ◽  
M.F. Schmid ◽  
P. Matsudaira ◽  
W. Chiu
Keyword(s):  
High Resolution ◽  
Binding Proteins ◽  
Actin Binding ◽  
Eukaryotic Cells ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Actin Bundle ◽  
Actin Bundles ◽  
3 Dimensional ◽  
Macromolecular Assembly

Actin is a protein found in all eukaryotic cells. In its polymerized form, the cells use it for motility, cytokinesis and for cytoskeletal support. An example of this latter class is the actin bundle in the acrosomal process from the Limulus sperm. The different functions actin performs seem to arise from its interaction with the actin binding proteins. A 3-dimensional structure of this macromolecular assembly is essential to provide a structural basis for understanding this interaction in relationship to its development and functions.

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