fibrinogen-page for fibrinogenolytic agent analysis in peanut worm homogenate

<meta charset="utf8"></head><body>Onthulling van fibrinogenolytische middelen in pindawormen met fibrinogeen-PAGE

In recent years, due to the continued rise of thrombotic diseases, thrombotic diseases have become a new major global health problem1. At present, antithrombotic drugs are classified into three groups: anti-platelet aggregation drugs, anticoagulants, and thrombolytic drugs. Among them, thrombolytic drugs, such as urokinase (UK), tissue plasminogen activator (tPA), etc., are the only clinically used drugs that can hydrolyze thrombus2. Meanwhile, more safe and effective thrombolytic drugs are being developed by the identification of novel thrombolytic agents3.
However, the identification of novel thrombolytic agents is time-consuming and labor-intensive, which mainly involves the separation/purification and characterization/checking stages. The former is to separate each component, and the latter is to display their fibrino(geno)lytic activities4,5. In previous studies, despite the fact we have successfully isolated a novel fibrino(geno)lytic enzyme (sFE) from the peanut worm (Sipunculus nudus) by affinity chromatography and fibrin(ogen) plate assay6,7,8, these processes are such time and labor-consuming. First, it is necessary to determine whether the peanut worm homogenates have fibrino(geno)lytic activity by the fibrin(ogen) plate method and reaction products based on PAGE9. Then, a series of chromatography, such as ion exchange chromatography, gel filtration chromatography, affinity chromatography, and other methods, have to be carried out for separation and purification10,11. Subsequently, the fibrin plate assay is performed again to check the fibrinogenolytic activity of each separated component. Finally, native-PAGE and sodium dodecyl sulfate (SDS)-PAGE are performed to determine the molecular weight of the active fibrinogenolytic agents12. Therefore, it is critical to rapidly separate and display the active fibrinogenolytic agents.
To rapidly separate and display the active fibrinogenolytic agents in the peanut worm homogenates, a new fibrinogen-PAGE method was developed by combining the PAGE and fibrinogen plate methods, i.e., the substrates of fibrinogenolytic agents fibrinogen were added to the native-PAGE gel. After native-PAGE, the components were separated by their molecular weight. Meanwhile, each active fibrinogenolytic component can be displayed by staining. By this method, we successfully detected more than one active fibrinogenolytic agent of peanut worm&#160;homogenate within 6 h. Moreover, this fibrinogen-PAGE method is time and cost-friendly. Furthermore, this method could be used to study the fibrinogenolytic agents of the other organisms.

<meta charset="utf8"></head><body>Auteur in de schijnwerpers: Tijd- en kosteneffectief fibrinogeen-PAGE voor fibrinogenolytische studies

Snelle scheiding en weergave van actieve fibrinogenolytische middelen in Sipunculus nudus door middel van fibrinogeen-polyacrylamide-gelelektroforese

Our research is focused on the fibrinolytic system. In this study, we are trying to simplify the mass for screening of fibrinogenolytical agents in peanut worms using Fibrinogen-PAGE technique. We have developed a new Fibrinogen-PAGE method by combining the PAGE and Fibrinogen plate methods.Compared to the other technics currently used, our protocol consists of a 10 year study separate and display the active fibrinogenolytic genes within the same pathogen. Overall, this mass is time and cost effective. With our findings, the discovery of neuro-fibrinogenolytic agents will become simple and can speed up the development of novel drugs that can be used to treat a variety of cardiovascular and other diseases.Our laboratory will focus on studying the coagulation and the fibrinogenolytic systems in different organisms to isolate and screen for novel fibrinogenolytic agents that can be useful for therapeutics.

Fibrinogeen-PAGE voor analyse van fibrinogenolytische middelen in pindawormhomogenaat

fibrinogen-page-for-fibrinogenolytic-agent-analysis-peanut-worm

Research

JoVE Journal

Biochemistry

Fibrinogen-PAGE for Fibrinogenolytic Agent Analysis in Peanut Worm Homogenate

39 Views. To begin, prepare a gel for fibrinogen phage and place it in the electrophoresis tank together with the glue mold. Pour 1, 300 milliliters of electrophoresis solution into the tank and take out the comb from the gel. Mix 5 microliters of 5x non-denaturing loading buffer with 20 microliters each of fibrinogenolytic enzyme and peanut worm homogenate.Load the mixture into the prepared gel and initiate the electrophoresis at 80 volts for 30 minutes or 120 volts for 20 minutes. Remove the g...

Video: Fibrinogen-PAGE for Fibrinogenolytic Agent Analysis in Peanut Worm Homogenate

fabricating van der waals heterostructures with precise rotational alignment

Fabricating van der Waals Heterostructures with Precise Rotational Alignment

In this work we describe a technique that is used to create new crystals (van der Waals heterostructures) by stacking ultrathin layered 2D materials with precise control over position and relative...

rapid separation and display of active fibrinogenolytic agents in sipunculus nudus through fibrinogen-polyacrylamide gel electrophoresis

Author Spotlight: Time and Cost-Effective Fibrinogen-PAGE for Fibrinogenolytic Studies

Here, we present a fibrinogen-polyacrylamide gel electrophoresis (PAGE) protocol to rapidly separate and display the fibrinogenolytic agents of Sipunculus...

Unveiling Fibrinogenolytic Agents in Peanut Worms with Fibrinogen-PAGE

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multimer-page for separating native protein complexes: a hybrid separation technique consisting of blue native-page and sds-page to separate intact multimeric proteins from tissue lysate

This video describes multimeric PAGE for separating native protein complexes from tissue homogenates. The technique is a hybrid of blue native-PAGE and SDS-PAGE techniques. The separated complexes can be characterized and studied for their role in cell...

Multimer-PAGE for Separating Native Protein Complexes: A Hybrid Separation Technique Consisting of Blue Native-PAGE and SDS-PAGE to Separate Intact Multimeric Proteins From Tissue Lysate

sds-page/immunoblot detection of a&beta; multimers in human cortical tissue homogenates using antigen-epitope retrieval

SDS-PAGE/Immunoblot Detection of A&amp;#946; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the A&beta; peptide. Using this protocol, we consistently detect monomeric and multimeric A&beta; in cortical tissue from humans with Alzheimer's...

SDS-PAGE/Immunoblot Detection of A&beta; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

After disassembling the electrophoresis apparatus, separate the glass panes of the gel cassette. Remove and discard the stacking...

denaturing urea polyacrylamide gel electrophoresis (urea page)

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular...

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

multimer-page: a method for capturing and resolving protein complexes in biological samples

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is...

probe type ii band alignment in one-dimensional van der waals heterostructures using first-principles calculations

Probe Type II Band Alignment in One-Dimensional Van Der Waals Heterostructures Using First-Principles Calculations

Calculations performed by the Vienna Ab initio Simulation Package can be used to identify the intrinsic electronic properties of nanoscale materials and predict the potential water-splitting...

separation of polymerized nucleic acid constructs by page 

Separation of Polymerized Nucleic Acid Constructs by PAGE   

resolving affinity purified protein complexes by blue native page and protein correlation profiling

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein...

non-reducing sds page: a method to analyze disulfide-linked multimeric protein complexes

This video describes the technique of separating the protein samples by non-reducing SDS-PAGE to analyze the multimeric protein complexes. This technique retains the multimer subunits of a protein held by the disulfide bonds which can later be analyzed by western...

Non-Reducing SDS PAGE: A Method to Analyze Disulfide-Linked Multimeric Protein Complexes

isolating mouse tear protein and its analysis using sds-page

Isolating Mouse Tear Protein and its Analysis Using SDS-PAGE

preparing peanut worm homogenate and gel for fibrinogen-page to identify fibrinogenolytic agents

Preparing Peanut Worm Homogenate and Gel for Fibrinogen-PAGE to Identify Fibrinogenolytic Agents

Open the 16% pre-cast TGX SDS-PAGE package per the manufacturer&#39;s protocol, and remove the cassette. Remove the comb that is lining the wells, and the tape from the bottom of the cassette, and place the gel into the running apparatus. Fill the chamber with the 1X running buffer, until the wells are submerged in...

urea-polyacrylamide gel electrophoresis (page) for probing dna enzyme activities employing commercial oligonucleotides

Urea-Polyacrylamide Gel Electrophoresis (PAGE) for Probing DNA Enzyme Activities Employing Commercial Oligonucleotides

rab10 phosphorylation detection by lrrk2 activity using sds-page with a phosphate-binding tag

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase...

phloem sap sampling from brassica napus for 3d-page of protein and ribonucleoprotein complexes

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

Here we present a protocol to analyze the protein composition of large native protein:protein and protein:nucleic acid complexes from oilseed rape (B. napus) phloem exudate using a 3D polyacrylamide gel electrophoresis (PAGE) approach combining blue native (BN) with two denaturing PAGEs followed by mass spectrometric...

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

examining proteasome assembly with recombinant archaeal proteasomes and nondenaturing page:  the case for a combined approach

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach

This protocol uses both subunit coexpression and postlysis subunit mixing for a more thorough examination of recombinant proteasome...

combining non-reducing sds-page analysis and chemical crosslinking to detect multimeric complexes stabilized by disulfide linkages in mammalian cells in culture

Combining Non-reducing SDS-PAGE Analysis and Chemical Crosslinking to Detect Multimeric Complexes Stabilized by Disulfide Linkages in Mammalian Cells in Culture

Disulfide linkages have long been known to stabilize the structure of many proteins. A simple method to analyze multimeric complexes stabilized by these linkages is through non-reducing SDS-PAGE analysis. Here, this method is illustrated by analyzing the nuclear isoform of dUTPase from the human bone osteosarcoma cell line U-2 OS.

Rapid Separation and Display of Active Fibrinogenolytic Agents in Sipunculus nudus through Fibrinogen-Polyacrylamide Gel Electrophoresis

Fibrinogenolytic agents that can dissolve fibrinogen directly have been widely used in anti-coagulation treatment. Generally, identifying new fibrinogenolytic agents requires the separation of each component first and then checking their fibrinogenolytic activities. Currently, polyacrylamide gel electrophoresis (PAGE) and chromatography are mostly used in the separating stage. Meanwhile, the fibrinogen plate assay and reaction products based PAGE are usually adopted to display their fibrinogenolytic activities. However, because of the spatiotemporal separation of those two stages, it is impossible to separate and display the active fibrinogenolytic agents with the same gel. To simplify the separating and displaying processes of fibrinogenolytic agent identification, we constructed a new fibrinogen-PAGE method to rapidly separate and display the fibrinogenolytic agents of peanut worms (Sipunculus nudus)&#160;in this study. This method includes fibrinogen-PAGE preparation, electrophoresis, renaturation, incubation, staining, and decolorization. The fibrinogenolytic activity and molecular weight of the protein can be detected simultaneously. According to this method, we successfully detected more than one active fibrinogenolytic agent of peanut wormhomogenate within 6 h. Moreover, this fibrinogen-PAGE method is time and cost-friendly. Furthermore, this method could be used to study the fibrinogenolytic agents of the other organisms.

Here, we present a fibrinogen-polyacrylamide gel electrophoresis (PAGE) protocol to rapidly separate and display the fibrinogenolytic agents of Sipunculus nudus.

Fibrinogenolytic agents that can dissolve fibrinogen directly have been widely used in anti-coagulation treatment. Generally, identifying new ...

To begin, weigh 50 grams of peanut worms and add them into the homogenizer, along with 150 milliliters of saline. Homogenize at 24, 000 RPM for 60 seconds. Centrifuge the homogenate and collect the supernatant for further processes.For fibrinogen polyacrylamide gel preparation, add 0.01 grams of fibrinogen and other reagents into a 50 milliliter glass beaker. Warm the beaker at 37 degrees Celsius for 30 minutes. After adding acrylamide, pour the separating gel mixture into a 10 well gel mold.Then add two milliliters of double distilled water to the gel mold and incubate it for 30 minutes. Invert the mold to remove the water. Next, prepare the loading gel similarly, and pour it onto the same mold as earlier.Finally, insert the comb for well formation.

Bereiding van pindawormhomogenaat en gel voor fibrinogeen-PAGE om fibrinogenolytische middelen te identificeren

To begin, prepare a gel for fibrinogen phage and place it in the electrophoresis tank together with the glue mold. Pour 1, 300 milliliters of electrophoresis solution into the tank and take out the comb from the gel. Mix 5 microliters of 5x non-denaturing loading buffer with 20 microliters each of fibrinogenolytic enzyme and peanut worm homogenate.Load the mixture into the prepared gel and initiate the electrophoresis at 80 volts for 30 minutes or 120 volts for 20 minutes. Remove the gel from the glue mold and transfer it into a plastic box. Add Tris-HCL and Triton into the box and incubate it on a shaker at 60 to 70 RPM for 10 minutes.Remove the wash buffer and incubate the gel with 50 milliliters of 0.05 molar Tris-HCL. After removing the buffer, incubate the gel with 50 milliliters of staining, followed by de-colorization solution at 60 to 70 RPM for 30 minutes each. The peanut worm homogenate displayed bands corresponding to molecular weights of 25, 33, 43, and 70 kilo-daltons or higher.