Studying Protein-Protein Interactions via Blot Overlay/Far Western Blot

2015 ◽  
pp. 371-379
Author(s):  
Randy A. Hall
Keyword(s):  
Western Blot ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Far Western Blot

scholarly journals Proteomic Analysis of Antigen 60 Complex of M. bovis Bacillus Calmette-Guérin Reveals Presence of Extracellular Vesicle Proteins and Predicted Functional Interactions

Vaccines ◽  
2019 ◽  
Vol 7 (3) ◽  
pp. 80 ◽  
Author(s):  
Khayriyyah Mohd Hanafiah ◽  
Norsyahida Arifin ◽  
Paul R. Sanders ◽  
Nurulhasanah Othman ◽  
Mary L. Garcia ◽  
...  
Keyword(s):  
Western Blot ◽  
Protein Interactions ◽  
Ribosomal Proteins ◽  
Vaccine Development ◽  
Extracellular Vesicle ◽  
Biological Interactions ◽  
Bacillus Calmette Guerin ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Antigen 60

Tuberculosis (TB) is ranked among the top 10 causes of death worldwide. New biomarker-based serodiagnostics and vaccines are unmet needs stalling disease control. Antigen 60 (A60) is a thermostable mycobacterial complex typically purified from Bacillus Calmette-Guérin (BCG) vaccine. A60 was historically evaluated for TB serodiagnostic and vaccine potential with variable findings. Despite containing immunogenic proteins, A60 has yet to be proteomically characterized. Here, commercial A60 was (1) trypsin-digested in-solution, analyzed by LC-MS/MS, searched against M. tuberculosis H37Rv and M. bovis BCG Uniprot databases; (2) analyzed using STRING to predict protein–protein interactions; and (3) probed with anti-TB monoclonal antibodies and patient immunoglobulin G (IgG) on Western blot to evaluate antigenicity. We detected 778 proteins in two A60 samples (440 proteins shared), including DnaK, LprG, LpqH, and GroEL1/2, reportedly present in mycobacterial extracellular vesicles (EV). Of these, 107 were also reported in EVs of M. tuberculosis, and 27 key proteins had significant protein–protein interaction, with clustering for chaperonins, ribosomal proteins, and proteins for ligand transport (LpqH and LprG). On Western blot, 7/8 TB and 1/8 non-TB sera samples had reactivity against 37–50 kDa proteins, while LpqH, GroEL2, and PstS1 were strongly detected. In conclusion, A60 comprises numerous proteins, including EV proteins, with predicted biological interactions, which may have implications on biomarker and vaccine development.

Characterization of group B streptococcal glyceraldehyde-3-phosphate dehydrogenase: surface localization, enzymatic activity, and protein–protein interactions

2003 ◽  
Vol 49 (5) ◽  
pp. 350-356 ◽  
Author(s):  
Kyle N Seifert ◽  
William P McArthur ◽  
Arnold S Bleiweis ◽  
L Jeannine Brady
Keyword(s):  
Monoclonal Antibody ◽  
Western Blot ◽  
Protein Interactions ◽  
Fluid Phase ◽  
Protein Protein Interactions ◽  
Group B Streptococci ◽  
Whole Cells ◽  
Surface Localization ◽  
Group B

During characterization of the surface antigens of serotype III group B streptococci (GBS), a protein with an apparent Mr~ 173 500 migrating on a SDS – polyacrylamide gel was found to have an N-terminal amino acid sequence identical to that of the plasmin receptor (Plr) of group A streptococci, a surface-localized glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This work begins to characterize GBS GAPDH and to assess its functional activity on the cell surface. The 1.0-kb gapC gene of GBS was amplified by PCR. plr and gapC demonstrated 87% homology. An anti-Plr monoclonal antibody reacted with GBS whole cells, suggesting GBS GAPDH is surface localized. Multiple serotypes of GBS demonstrated functional GAPDH on their surfaces. The anti-Plr monoclonal antibody recognized GBS protein bands of approximately 41 and 173.5 kDa, by Western blot. Presumably, these represent monomeric and tetrameric forms of the GAPDH molecule. GBS GAPDH was demonstrated by Western blot analysis to interact with lys- and glu-plasminogens. Fluid-phase GBS GAPDH interacted, by means of ELISA, with immobilized lys-plasminogen, glu-plasminogen, actin, and fibrinogen. Enzymatically active GAPDH, capable of binding cytoskeletal and extracellular matrix proteins, is expressed on the surface of GBS.Key words: group B streptococci, glyceraldehyde-3-phosphate dehydrogenase.

Protein Expression Analysis by Western Blot and Protein–Protein Interactions

2019 ◽  
pp. 101-111
Author(s):  
María Dolores Cima-Cabal ◽  
Fernando Vazquez ◽  
Juan R. de los Toyos ◽  
María del Mar García-Suárez
Keyword(s):  
Protein Expression ◽  
Western Blot ◽  
Expression Analysis ◽  
Protein Interactions ◽  
Protein Protein Interactions

scholarly journals Mechanistic insight into light-dependent recognition of Timeless by Drosophila cryptochrome

2021 ◽  
Author(s):  
Changfan Lin ◽  
Connor M. Schneps ◽  
Siddarth Chandrasekaran ◽  
Abir Ganguly ◽  
Brian R. Crane
Keyword(s):  
Molecular Dynamics ◽  
Western Blot ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Esr Spectroscopy ◽  
Redox State ◽  
Protein Protein Interactions ◽  
Mechanistic Insight ◽  
Dynamics Simulations ◽  
Insight Into

SUMMARYCryptochrome (CRY) entrains the fly circadian clock by binding to Timeless (TIM) in light and triggering its degradation. Undocking of a helical C-terminal tail (CTT) in response to photoreduction of the CRY flavin cofactor gates TIM binding. A generally-applicable Select Western-blot-Free Tagged-protein Interaction (SWFTI) assay enables quantification of CRY binding to TIM in dark and light. The assay is utilized to study CRY variants with residue substitutions in the flavin pocket and correlate their TIM affinities with CTT undocking, as measured by pulse-dipolar ESR spectroscopy and evaluated by molecular dynamics simulations. CRY variants with the CTT removed or undocked bind TIM constitutively, whereas those incapable of photoreduction bind TIM weakly. In response to flavin redox state, two conserved histidine residues contribute to a robust on/off switch by mediating CTT interactions with the flavin pocket and TIM. Our approach provides an expeditious means to quantify protein-protein interactions and photoreceptor targeting.

Protein-protein interactions of the p53 family with the Bcl-2 family in hepatocellular carcinoma

2011 ◽  
Vol 49 (08) ◽  
Author(s):  
LC König ◽  
M Meinhard ◽  
C Sandig ◽  
MH Bender ◽  
A Lovas ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
P53 Family

Partial Purification of a Specific Plasminogen Activator from Porcine Parotid Glands

1974 ◽  
Vol 31 (03) ◽  
pp. 403-414 ◽  
Author(s):  
Terence Cartwright
Keyword(s):  
Nucleic Acid ◽  
Salivary Glands ◽  
Plasminogen Activator ◽  
Protein Interactions ◽  
Partial Purification ◽  
Protein Protein Interactions ◽  
Parotid Glands ◽  
Two Stage ◽  
Preliminary Characterization ◽  
Specific Inhibitors

SummaryA method is described for the extraction with buffers of near physiological pH of a plasminogen activator from porcine salivary glands. Substantial purification of the activator was achieved although this was to some extent complicated by concomitant extraction of nucleic acid from the glands. Preliminary characterization experiments using specific inhibitors suggested that the activator functioned by a similar mechanism to that proposed for urokinase, but with some important kinetic differences in two-stage assay systems. The lack of reactivity of the pig gland enzyme in these systems might be related to the tendency to protein-protein interactions observed with this material.

Target-Templated de novo Design of Macrocyclic D-/L-Peptides: Inhibitors of the PD-1/PD-L1 Interaction

2020 ◽  
Author(s):  
Salvador Guardiola ◽  
Monica Varese ◽  
Xavier Roig ◽  
Jesús Garcia ◽  
Ernest Giralt
Keyword(s):  
Protein Interactions ◽  
Cyclic Peptides ◽  
General Framework ◽  
Large Scale ◽  
De Novo ◽  
Inhibitory Effect ◽  
Original Text ◽  
Protein Protein Interactions ◽  
Retraction Notice ◽  
Pharmaceutical Properties

<p>NOTE: This preprint has been retracted by consensus from all authors. See the retraction notice in place above; the original text can be found under "Version 1", accessible from the version selector above.</p><p><br></p><p>------------------------------------------------------------------------</p><p><br></p><p>Peptides, together with antibodies, are among the most potent biochemical tools to modulate challenging protein-protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing target-specific binders with improved pharmaceutical properties, such as macrocyclic peptides. Here we report a general framework that leverages the computational power of Rosetta for large-scale backbone sampling and energy scoring, followed by side-chain composition, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we identified two peptides (PD-<i>i</i>3 and PD-<i>i</i>6) that target PD-1, a key immune checkpoint, and work as protein ligand decoys. A comprehensive biophysical evaluation confirmed their binding mechanism to PD-1 and their inhibitory effect on the PD-1/PD-L1 interaction. Finally, elucidation of their solution structures by NMR served as validation of our <i>de novo </i>design approach. We anticipate that our results will provide a general framework for designing target-specific drug-like peptides.<i></i></p>

Sign in / Sign up

Export Citation Format

Share Document