bacterial culture filtrate protein concentration using an ultrafiltration device

Bacterial Culture Filtrate Protein Concentration using an Ultrafiltration Device

To begin, prepare a 100 kilodalton molecular weight cutoff centrifugal filter by adding the full volume capacity of PBS. Centrifuge for five minutes at 2,800 times g at 4 degrees Celsius. Discard the flow through, and any remaining PBS, and fill the sample chamber with Mtb CFP to its maximum capacity. Centrifuge at 2,800 times g at 4 degrees Celsius until the volume has reduced to the minimum volume of the ultrafiltration device. Repeat as necessary until the entire sample has been reduced sufficiently.
Add 1x PBS to the filter unit containing the concentrate up to the total device capacity. Reduce the volume as described before and repeat this step five times to ensure complete washing and buffer exchange. Then, recover the 100 kilodalton-concentrated CFP retentate, or 100R, according to the ultrafiltration device specifications. Wash the filter with a minimal volume of 1x PBS at least three times, and pull the wash with the 100R to maximize the recovery.

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1. Preparation of crude Mycobacterium tuberculosis (Mtb) extracellular vesicle (EV) concentrate
NOTE: For detailed procedures on the cultivation of Mtb and preparation of culture filtrate protein (CFP). It is recommended that bacterial culture media is free from growth supplements with EV-containing or proteinaceous components, such as Oleic Albumin Dextrose Catalase (OADC), and detergents such as Tween. It is also recommended that the bacterial culture&#39;s quality and harvested CFP be screened to ensure limited cell death and lysis.
<ol>
	<li>Prepare a 100 kDa molecular weight cut-off (MWCO) centrifugal filter (see Table of Materials) by adding the full volume capacity of phosphate-buffered saline (1x PBS). Centrifuge for 5 min at 2,800 x g at 4 &#176;C. 
	NOTE: If using a filter with no dead stop volume, care must be taken to ensure the sample volume does not reduce below the filter level, resulting in complete filter drying during centrifugation.</li>
	<li>Discard the flow-through and any remaining PBS before filling the sample chamber with Mtb CFP to its maximum capacity. Centrifuge at 2,800 x g at 4 &#176;C until the volume has reduced to the minimum volume of the ultrafiltration device. Repeat as necessary, adding more CFP to the unit until the entire sample has been reduced sufficiently. 
	NOTE: Retain a portion of the 100 kDa flow-through (100F) material for downstream qualification if desired. Store at 4 &#176;C.</li>
	<li>Add 1x PBS to the filter unit containing the concentrate up to the total device capacity. Reduce the volume as described in 1.2. Repeat this step five times to ensure complete washing and buffer exchange.</li>
	<li>Recover the 100 kDa concentrated CFP retentate (100R) according to the ultrafiltration device specifications (see Table of Materials). Once the 100R is recovered, wash the filter with a minimal volume of 1x PBS at least three times, and pool the wash with the 100R to maximize the recovery.</li>
	<li>Quantitate the 100R material with a bicinchoninic assay (BCA) following the manufacturer&#39;s instructions (see Table of Materials). To perform the assay, use several dilutions of samples 1:2, 1:5, and 1:10 in PBS. Test the sample in triplicate. 
	NOTE: Retain a portion of the 100R material for downstream qualification if desired. Store at 4 &#176;C.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Automatic Fraction Collector</td>
			<td>IZON Science</td>
			<td>AFC-V1-USD</td>
			<td></td>
		</tr>
		<tr>
			<td>Centricon Plus - 70 Centrifugal filter, 100 kDa cutoff</td>
			<td>Millipore Sigma</td>
			<td>UFC710008</td>
			<td>Ultrafiltration device used in step 1.1</td>
		</tr>
		<tr>
			<td>Phosphate-buffered Saline, 1X without calcium and magnesium</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane, Roll, 0.2 &#956;m</td>
			<td>BioRad</td>
			<td>1620112</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Ryan, J. M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63895">Mycobacterium tuberculosis</a><a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63895"> Extracellular Vesicle Enrichment through Size Exclusion Chromatography.</a> J. Vis. Exp. (183) (2022)
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.

1. Preparation of crude Mycobacterium tuberculosis (Mtb) extracellular vesicle (EV) concentrate
NOTE: For detailed procedures on the cultivation of Mtb ...

Take a large-volume centrifugal filter device containing vertical regenerated cellulose membranes of a suitable pore size.
Add buffer and centrifuge. Remove the flow-through containing membrane humectant.
Load the device with bacterial culture filtrate protein, or CFP, encompassing soluble proteins and extracellular vesicles, or EVs &#160;&#8211;&#160; nanosized lipid-bound structures containing biomolecules secreted by bacteria.
During centrifugation, CFP passes tangentially across the membranes via centrifugal force.
Biomolecules smaller than the membrane&#39;s pores pass through with culture fluid, while larger ones, such as EVs, pass over the membrane surface, minimizing membrane clogging.
The filter concentrates the CFP into a minimum device volume, aided by its dead-stop volume to prevent biomolecule drying.
Add buffer and centrifuge.
The buffer replaces residual media components, creating an isotonic environment for CFP biomolecules and maintaining their integrity.
Place the concentrate cup over the filter cup, invert, and centrifuge. Recover the concentrated CFP retentate for further analysis.

70 Aufrufe. Filtratproteinkonzentration in Bakterienkulturen mit einem Ultrafiltrationsgerät

Serie: Filtratproteinkonzentration in Bakterienkulturen mit einem Ultrafiltrationsgerät - Experiment

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Modellierung von Tuberkulose Mycobacterium Marinum infizierten Erwachsenen Zebrafisch

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

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Immunologie und Infektion

Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) containing immunologically active molecules. Knowledge regarding the molecular mechanisms of vesicle biogenesis, the content of the vesicles, and their functions at the pathogen-host interface is very limited. Addressing these questions requires rigorous procedures for isolation, purification, and validation of EVs. Previously, vesicle production was found to be enhanced when M. tuberculosis was exposed to iron restriction, a condition encountered by Mtb in the host environment. Presented here is a complete and detailed protocol to isolate and purify EVs from iron-deficient mycobacteria. Quantitative and qualitative methods are applied to validate purified EVs.

Mycobacterium tuberculosis shows increased production and release of extracellular vesicles in response to low iron conditions. This work details a protocol for generating low iron conditions and methods for the purification and characterization of mycobacterial extracellular vesicles released in response to iron deficiency.

Isolierung und Charakterisierung von extrazellulären Vesikeln, die von eisenbegrenzten Mykobakterien hergestellt werden

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) ...

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

Synthese, Charakterisierung und Anwendung von superparamagnetischen Eisenoxid-Nanosonden zur Erkennung von extrapulmonaler Tuberkulose

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

Bacterial Culture Filtrate Protein Concentration using an Ultrafiltration Device

Transkript

Bacterial Culture Filtrate Protein Concentration using an Ultrafiltration Device