Name: Scheiding en fractionering van kweekfiltraateiwitten (CFPs) van Mycobacterium tuberculosis (Video) | JoVE Journal
Uploaded: 2025-07-11T12:50:00.000+00:00
Duration: 8 min 48 s
Description: Leer de scheiding van kweekfiltraateiwitten van Mycobacterium tuberculosis met isoelectrische focus en SDS-PAGE. Deze methode identificeert immunogene fracties voor diagnostiek en vaccinontwikkeling.

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scheiding en fractionering van kweekfiltraateiwitten (cfp's) van mycobacterium tuberculosis

Separation and Fractionation of Culture Filtrate Proteins (CFPs) from Mycobacterium tuberculosis

Scheiding en fractionering van kweekfiltraateiwitten (CFP's) van Mycobacterium tuberculosis

Secreted proteins of Mycobacterium tuberculosis (M. tuberculosis) play a crucial role in tuberculosis pathogenesis, immune modulation, and disease ...

separation-fractionation-culture-filtrate-proteins-cfps-from

Research

JoVE Journal

Immunology and Infection

Immunologie en Infectie

Source: Gupta, S., Marcela Rodriguez, G. Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria. J. Vis. Exp. (2019)This video demonstrates the stepwise isolation of extracellular vesicles (EVs) from Mycobacterium tuberculosis cultured under iron-limiting conditions that mimic the host environment. It outlines the use of ultrafiltration, differential centrifugation, and density gradient separation to enrich and identify EV-containing fractions.

Isolation of Siderophore-Containing Extracellular Vesicles from Mycobacterium tuberculosis

This video demonstrates the thin-layer chromatography-based separation of mycolic acids. This technique separates the different mycolic acids—long-chain lipids present in the cell wall of mycobacteria—based on their polarity.

Thin Layer Chromatography-Based Separation of Mycolic Acid Variants: A Method to Separate Mycobacterial Cell Wall Lipids Based on Polarity

This video demonstrates the isolation and concentration of bacterial culture filtrate proteins, which include bacterial extracellular vesicles, using ultrafiltration. The concentrated extracellular vesicles serve as diagnostic markers for disease detection.

Bacterial Culture Filtrate Protein Concentration using an Ultrafiltration Device

This video demonstrates the size exclusion chromatography (SEC) technique for purifying bacterial extracellular vesicles (EVs) from the culture filtrate of Mycobacterium tuberculosis (Mtb). The technique utilizes a porous resin-packed column to obtain purified fractions of EVs. The porous particles selectively allow smaller proteins to enter the pores, resulting in slower elution. At the same time, EVs are excluded from entering the pores due to their larger size and are eluted first.

A Size Exclusion Chromatography Technique to Purify Mycobacterial Extracellular Vesicles

Het doel is om bioactieve kleine moleculen, peptiden uit een complex plantenextract en eiwitten uit pathogene microben te fractioneren en te isoleren door gebruik te maken van de isoelectrische focusmethode (IEF) met vloeibare fase. Verder werden de gescheiden moleculen geïdentificeerd en hun bioactiviteit bevestigd.

Scheiding van bioactieve kleine moleculen, peptiden uit natuurlijke bronnen en eiwitten uit microben door preparative isoelectric Focus (IEF) Methode

Snelle en efficiënte kwantificering van intracellulaire M. tuberculosis-groei is cruciaal voor het nastreven van verbeterde therapieën tegen tuberculose (TB). Dit protocol beschrijft een op bouillon gebaseerde colorimetrische detectietest met behulp van een geautomatiseerd vloeistofkweeksysteem om de Mtb-groei te kwantificeren in macrofagen die worden behandeld met kandidaat-gastheergerichte therapieën.

Een geautomatiseerd kweeksysteem voor gebruik bij preklinische tests van gastheergerichte therapieën voor tuberculose

Bacteriën scheiden nanometergrote extracellulaire blaasjes (EV's) af die bioactieve biologische moleculen dragen. EV-onderzoek richt zich op het begrijpen van hun biogenese, rol in microbe-microbe en gastheer-microbe interacties en ziekte, evenals hun potentiële therapeutische toepassingen. Een workflow voor schaalbare isolatie van EV's van verschillende bacteriën wordt gepresenteerd om standaardisatie van EV-onderzoek te vergemakkelijken.

Schaalbare isolatie en zuivering van extracellulaire blaasjes van Escherichia coli en andere bacteriën

Dit protocol beschrijft grootte-uitsluitingschromatografie, een gemakkelijke en reproduceerbare techniek voor het verrijken van Mycobacterium tuberculosis extracellulaire blaasjes uit kweeksupernatanten.

Mycobacterium tuberculose Extracellulaire vesikelverrijking door grootte-uitsluitingschromatografie

Dit protocol beschrijft de verrijking van inheemse mycobacteriële extracellulaire vesikels (mEV's) uit axenische culturen van Mycobacterium smegmatis (Msm) en hoe mCherry (een rode fluorescerende reporter)-bevattende recombinante MsmEV's kunnen worden ontworpen en verrijkt. Ten slotte verifieert het de nieuwe aanpak met de verrijking van MsmEV's die het EsxA-eiwit van Mycobacterium tuberculosis bevatten.

Verrijking van inheemse en recombinante extracellulaire blaasjes van mycobacteriën

Hier demonstreren we de bereiding  van Mtb-cultuurfiltraat (CF), het bacteriegroeimedium dat bestaat uit eiwitten die door de bacteriën worden uitgescheiden. Mtb scheidt eiwitten af om gastheersignalering en immuunrespons te moduleren, die cruciaal zijn voor pathogenese. De bereide CF's worden geanalyseerd met behulp van massaspectrometrie om alle secretoire factoren te identificeren.

Bereiding van Mycobacterium tuberculosis Kweekfiltraat om de pathogenese van tuberculose te begrijpen

Dit protocol scheidt onoplosbare celwand- en membraaneiwitten in eenvoudige fracties (1-5 eiwitten) met behulp van preparatieve iso-elektrische focussering (IEF) op basis van iso-elektrisch punt, gevolgd door scheiding op molecuulgewicht. De resulterende fracties kunnen direct worden gebruikt voor immunologische en proteomische analyse zonder verdere zuivering.

Scheiding en fractionering van celwand- en celmembraaneiwitten van Mycobacterium tuberculosis voor stroomafwaartse eiwitanalyse

Leer de scheiding van kweekfiltraateiwitten van Mycobacterium tuberculosis met isoelectrische focus en SDS-PAGE. Deze methode identificeert immunogene fracties voor diagnostiek en vaccinontwikkeling.

Scheiding en fractionering van kweekfiltraateiwitten (CFP's) van Mycobacterium tuberculosis

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