This document outlines an experiment that used FRET microscopy to investigate the dynamics of the Z-ring in E. coli cells. The experiment used E. coli strains containing both FtsZ-CFP and FtsZ-YFP to observe FRET signals during cell division. Results showed donor, acceptor, and FRET signals over time, but the patterns did not match expectations, indicating issues with the experimental or analysis methods. Improvements discussed including changing inducer concentration and wait time before microscopy, as well as improving the image analysis methodology.
A Beginner’s Guide to the Principles and Applications of FRETExpedeon
FRET, or Fluorescence Resonance Energy Transfer, was first described more than 50 years ago. The availability of new dyes and detection technology has resulted in a much wider use of the application in recent years, especially in biomedical R&D.
A Beginner’s Guide to the Principles and Applications of FRETExpedeon
FRET, or Fluorescence Resonance Energy Transfer, was first described more than 50 years ago. The availability of new dyes and detection technology has resulted in a much wider use of the application in recent years, especially in biomedical R&D.
It is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels used as support media.
Gels are made by free radical-induced polymerization of acrylamide and N,N’-Methylenebisacrylamide.
It is the most widely used technique of electrophoresis.
Peptide Mass Fingerprinting (PMF) and Isotope Coded Affinity Tags (ICAT)Suresh Antre
Analytical technique for identifying unknown protein. The peptide mass are compared to database containing the theoretical peptide masses of all known protein sequences.
Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.
The next generation sequencing platform of roche 454creativebiogene1
454 is totally different from Solexa and Hiseq of Illumina. The disadvantage of 454 is that it is unable to accurately measure the homopolymer length. For this unavoidable reason, 454 technology will introduce insertion and deletion sequencing errors to the results.
Isoelectric focusing electrophoresis- Principle , procedure and applicationsJaskiranKaur72
IEF separates amphoteric compounds, such as proteins, with increased resolution in a medium possessing a stable pH gradient. The protein becomes “focused” at a point on the gel as it migrates to a zone where the pH of the gel matches the protein's pI. At this point, the charge of the protein becomes zero and its migration ceases.
It is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels used as support media.
Gels are made by free radical-induced polymerization of acrylamide and N,N’-Methylenebisacrylamide.
It is the most widely used technique of electrophoresis.
Peptide Mass Fingerprinting (PMF) and Isotope Coded Affinity Tags (ICAT)Suresh Antre
Analytical technique for identifying unknown protein. The peptide mass are compared to database containing the theoretical peptide masses of all known protein sequences.
Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.
The next generation sequencing platform of roche 454creativebiogene1
454 is totally different from Solexa and Hiseq of Illumina. The disadvantage of 454 is that it is unable to accurately measure the homopolymer length. For this unavoidable reason, 454 technology will introduce insertion and deletion sequencing errors to the results.
Isoelectric focusing electrophoresis- Principle , procedure and applicationsJaskiranKaur72
IEF separates amphoteric compounds, such as proteins, with increased resolution in a medium possessing a stable pH gradient. The protein becomes “focused” at a point on the gel as it migrates to a zone where the pH of the gel matches the protein's pI. At this point, the charge of the protein becomes zero and its migration ceases.
“N2MO provides cutting-edge ex vivo insect based screening models for predicting ADMET properties of chemical substances in the early drug discovery phase. Insect models represents excellent model systems due to biological active test situation, combined with fast reproducibility with no need for resynthesizing compound and at same time the model produces data that are reflecting mammalian ADMET properties”
WEBINAR Characterisation of human pluripotent stem cells (ESCs and IPSC) and ...Quality Assistance s.a.
Valérie DEFFONTAINE, R&D Scientist, Quality Assistance
Webinar held on 8th June 2017.
The discovery of human pluripotent stem cells 10 years ago turned the spotlight on the potential of pluripotent stem cells for personalised cell therapy. The scientific interest then quickly shifted towards the use of these cells for safety pharmacology, drug discovery and disease modelling. For all these purposes, in the mid to long term, properly characterised cell banks will be necessary.
The characterisation of embryonic (ESC) and induced pluripotent stem cells (IPSC) used for manufacturing requires the development and validation of analytical methods (e.g. flow cytometry, microscopy, QPCR and bioassays). Cell characterisation includes the testing of cell product identity, determination of impurities, and assessment of biological activity and viability. Among the techniques available, flow cytometry is widely used to assess the expression of cell markers. Our laboratory has developed flow cytometry panels dedicated to the characterisation of extracellular and intracellular markers of ESC and IPSC, and to the detection of cell-related impurities. We proposed a method for the validation of flow cytometry panels according to the recommendations of international guidelines on the validation of analytical methods.
IPSC differentiated into cardiomyocytes and MSC-like cells were also used to test the performance of our flow cytometry panels to accurately monitor the manufacturing process of cell products.
In addition to the technical tips, this webinar aims at presenting a critical view on the use of flow cytometry platform for cell characterisation.
For more information, visit http://www.quality-assistance.com/analytical-services/CBMPs
Next generation-sequencing.ppt-convertedShweta Tiwari
The advance version, sequences the whole genome efficiently with high speed and high throughput sequencing at reduce cost is termed as Next Generation Sequencing (NGS) or massively parallel sequencing (MPS).
BLO: Transferring the macromolecule from gel to membrane followed by detection on the membrane using antibody is k/a blotting
molecular methods used to identify and measure specific DNA, RNA and protein in complex biological mixtures.
It is the technique för
transferring DNA, RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis.
IMMUNO BLOTTING:
Immunoblotting techniques use antibodies to identify target proteins .
They involve identification of protein target via antigen-antibody (or protein-ligand) specific reactions.
The Southern blot is used for transferring DNA,.
The Northern blot for RNA
The western blot for PROTEIN.
The Eastern blot for PROTEIN, post-translational modifications (PTMS) .
WESTERN BLOTTING:
Principle:
Western blotting technique is used for identification of particular protein from the mixture of protein.
In this method labelled antibody against particular protein is used identify the desired protein, so it is a specific test.
Western blotting is also known as immunoblotting because it uses antibodies to detect the protein.
METHODOLOGY:
Extraction of protein
2. Gel electrophoresis: SDS PAGE
3. Blotting: electrical or capillary blotting
4. Blocking: BSA
5. Treatment with primary antibody
6. Treatment with secondary antibody( enzyme labelled anti Ab)
7. Treatment with specific substrate; if enzyme is alkaline phosphatase, substrate is p-nitro phenyl phosphate which give color.
Streamlined next generation sequencing assay development using a highly multi...Thermo Fisher Scientific
Next generation sequencing (NGS) assay development for solid tumor sequencing requires characterization of variant calling directly from formalin-fixed paraffin embedded (FFPE) tissue samples. However, cell line based FFPE and human FFPE samples only contain 2 to 20 variants, which require laboratories to invest significant resources in sample sourcing and preparation when developing assays to detect 100+ variants
27. DISCUSSION
We have another strain of bacteria we use that is
only FtsZ-CFP (PZW36)
We have another strain of bacteria we use that is
only FtsZ-YFP (PZW79)
28. SOURCES
Quantitation of Protein-Protein Interaction, Methods
In Cell Biology, A. Periasamy (2008)
Robust Growth of Escherichia Coli, Current Biology,
P. Wang (2010)
Direct interactions of early and late assembling
division proteins in Escherichia coli cells resolved
by FRET, Molecular Microbiology, S. Alexeeva
(2010)
Binary Fission is a vegetative cell division that bacteria cells undergo. During this division FtsZ filaments, positioned in the Z-ring, has an orientation that is unknown.
Forster (or Fluorescent) Resonance Energy Transfer (FRET) is an effective technique to observe proteins in a specimen. FRET uses the spectral overlap of donor and acceptor fluorophores.
PDMS made surface of microchannel go from hydrophobic to hydrophilic.
10mg/ml BSA at 1.0ml/hr. was injected into microchannel after being diluted in 18ml of water.
A short needle with one end sealed by silicone to seal the inlet and outlet. Mount whole PDMS device onto aluminum sheet. Carefully mount device on edge of spinner in centrifuge. Centrifuge at 5000rpm for 10 min.
Add 5ml LB + 100mg/ml BSA medium + 125ul sodium salicylate +5ul (1M) IPTG. Inject fresh LB medium at 90 ul/hr. until cells are washed out.
Turn ON live cell box. Wait until cells divide exponentially (after 1-2 hrs.). Move stuff to IIC. Set Proper flow rate (about 30-90ul/hr.) which can take newly born cells away.
Select positions including at least 100cells, set up parameters for autofocus.
