Technical overview IPG and 2D. Rehydrating the IPG strip Loaded sample Applied IPG strip Overlay with mineral oil-prevents Drying of the strip Crystallization.

Slides:

Advertisements

Similar presentations

The first-dimension was performed in two steps. The first step consisted of hydrating the IPG strip (remove the thin cover strips from over the gel and.

Advertisements

Day 2 Morning. Comparative/Functional Proteomics.

Western Analysis Laboratory procedure that allows you to:

Gel electrophoresis The gel electrophoresis method was developed in the late 1960's. It is a fundamental tool for DNA sequencing.

Lab 9. Human Mitochondrial Analysis using PCR and Electrophoresis Major Goals of this Experiment Isolate mitochondrial DNA (mtDNA) from cheek cells and.

Sodium dodecyl sulfate- Polyacrylamide gel electrophoresis (SDS-PAGE) Irene Goh Rosarine Metusela.

Gel Electrophoresis Gel Electrophoresis.

Gel electrophoresis Ashti Mohammad Amin M.Sc. Molecular Biology Medical Research Center Hawler Medical University

BRIDGES 2014 Agarose Gel Visualization of Restriction Enzyme Digest.

Biotech Lab #5 DNA Goes to the Races “Gel electrophoresis”

By: Grace Forster and Ashlyn Cowan.  a process that seperates large molecules (including nucleic acids or proteins) by size, electric charge, and physical.

Comparative Proteomics Kit II: Western Blot Module

SEPARATION OF DNA FRAGMENTS BY LENGTH. Organic molecules such as DNA are charged. DNA is negatively charged because the phosphates (red circles) that.

Proteomics Day 2 Tech Talk. Activities 1.Run 1 dimension SDS-PAGE gel of proteins isolated on day one and stain with Coomassie Blue 2.Rehydrate protein.

“Gel electrophoresis”. Gel electrophoresis is a procedure for separating a mixture of molecules through a stationary material (gel) in an electrical field.

ENDURO Horizontal Electrophoresis Systems. Casting a Gel Attach a buffer dam to one side of gel tray Attach the other buffer dam to the other side of.

Prepered by:- Rana Al-Turki

Salk Institute Mobile Lab Gel Electrophoresis Teacher Instructions BioRad Set Up 8 groups Grossmont Kit.

LAB SIX Molecular Biology. Three samples of phage lambda DNA: 1.Digested with EcoRI 2.Digested with HindIII (fragments are of known size) 3.Undigested.

Chapter 10: Analysis of proteins. Purification schemes: 1. soluble recombinant proteins 2. insoluble recombinant proteins that are produced as inclusion.

Section B: Protein StructureYang Xu, College of Life Sciences Section B Protein Structure B2 Protein structure B3 Protein Analysis.

DNA Analysis Using Agarose Gel Electrophoresis Day 1

Molecular Weight Determination

Quality Control of Product

SDS-Polyacrylamide Gel Electrophoresis

Electrophoresis and 2D Gel Analysis

Collect Buccal Cells. PCR Polymerase Chain Reaction DNA/gene amplification.

Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass spectrometry for protein identification 2-Dimensional Gel Electrophoresis MALDI-TOF.

Cellulose Acetate Membrane Electrophoresis xiaoli Serum protein electrophoresis (CAE)

Comparative Proteomics Kit I: Protein Profiler Module

Qualitative Analysis of Product

Electrophoresis Electrophoresis is the movement of molecules by an electric current .This is practically done in a matrix to limit migration and contain.

Electrophoresis PAGE Dr Gihan Gawish.

Sodium DodecylSulphate- PolyAcrylamide Gel Electrophoresis (SDS-PAGE)

5. SEPARATION AND DETECTION OF PROTEINS II SDS-PAGE Jana Vobořilová, Anna Kotrbová-Kozak, Vlasta Fürstová, Tereza Kopská.

Overview of 2DE Complex mixture of proteins

Proteomics Terry Kotrla, MS, MT(ASCP)BB. Human Genome Project Launched 1990 took 13 years Estimated 100,000 human genes would be discovered Only 20,000-25,000.

Proteomics Module Day 1 Tech talk. Experiment: Yeast protein expression changes caused by H 2 O 2 exposure. ► 2 Control groups (A and B): nothing added.

Electrophoresis / SDS-PAGE

Analysis of DNA Fragments with Gel Electrophoresis

Agarose (Horizontal) Gel Electrophoresis Malasian word for seaweed is “agar-agar”. Agarose is derived from red seaweed. Electrophoresis means “carrying.

Separation of main plasma protein by using SDS-PAGE

SDS-Polyacrylamide Gel Electrophoresis

Preparing yeast cell extracts SDS-PAGE gives a snapshot of proteins in an extract Proteins are extracted from cells.

Report Draw a scheme of the GA20OX cloning procedure.

GEL ELECTROPHORESIS. FIRST, WHAT……  Simply put, gel electrophoresis is a technique used to separate molecules such a DNA, RNA and proteins according.

Gel Electrophoresis. Definition – COPY ME! Separation of DNA fragments according to size and charge Based on movement through a gel medium when an.

Gel electrophoresis is a method for separation and analysis of macromolecules(DNA, RNA and proteins) and their fragments, based on their size and charge.

Agarose gel electrophoresis. Agarose gel electrophoresis is an easy way to separate DNA fragments by their sizes and visualize them. It is a common diagnostic.

Sodium Dodecyl Sulfate-PolyacrylAmide gel Electrophoresis [SDS-PAGE] Experiment 7 BCH 333[practical]

Comparative Proteomics Kit II: Western Blot Analysis Module

Lecture-8 Introduction to Proteomics Huseyin Tombuloglu, Phd GBE423 Genomics & Proteomics.

DNA Fingerprinting Agarose Gel Electrophoresis Student Instructions

Gel Electrophoresis What is Gel Electrophoresis? Gel electrophoresis separates molecules on the basis of their charge and size. The charged macromolecules.

Comparative Proteomics Kit I: Protein Profiler Module

Proteomics Day 3 Tech Talk. Activities 1.Prepare IPG strip for second gel electrophoresis 2.Run IPG strip in second dimension SDS- PAGE electrophoresis.

Water – Part I CHEM 7784 Biochemistry Professor Bensley.

Enzyme Series Lab 4 SDS-PAGE Gel Electrophoresis.

Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass spectrometry for protein identification 2-Dimensional Gel Electrophoresis MALDI-TOF.

Gel Electrophoresis By: Sariah Arnold.

Two dimensional gel electrophoresis

Electrophoresis The purpose of electrophoresis is to separate molecules of DNA, RNA or protein. Separation can be based upon: Size Shape Isoelectric point.

Gel Electrophoresis Method of separating molecules within an electric field based on the size and charge of DNA fragments.

Agarose gel Electrophoresis

Agarose Gel Electrophoresis

Creating a DNA Fingerprint by Gel Electrophoresis

Last class Salting out Dialysis Paper 1 discussion.

Gel Electrophoresis 3/5/18.

Presentation transcript:

Technical overview IPG and 2D

Rehydrating the IPG strip Loaded sample Applied IPG strip Overlay with mineral oil-prevents Drying of the strip Crystallization of the urea Uptake of O2 and CO2 50 volts overnight UVM Proteomics Outreach Module

Turned off IEF apparatus Inserted pre-wet Wicks between electrode wire and IPG strip [both sides] Wicks are required to absorb salts during the IEF Turn on IEF apparatus on run the following cycle: 250 volts 15 minutes 8000 volts 2.5 hours Hold at 500 volts Next Day….. -20

Todays Lab: Running the 2D gel Basically running a standard SDS-PAGE Wash IPG strip in Equilibration buffer[s] (EB) containing: Urea-chaotropic salt- disrupts non-covalent bonds including hydrogen bonds Van der Waals Hydrophobic SDS binds to protein and makes negatively charged DTT Breaks disulfide bonds Iodoacetimide -Alkylating sulfhydryl molecule Binds to Cysteines Prevents Disulfide bridges

Protocol Remove excess oil from IPG strip Make sure EB1 and EB2 have their additives (DTT and Iodoacetamide) Add EB1 buffer to strip [s] and rotate for 10 minutes Transfer to EB2 and rotate for 10 minutes Prepare the Bio-Rad PreCast Gel…..peel strips Install IPG strip in the precast gel….+ end to the left!!! Overlay the strip with melted agarose Load standard and cover that with Agarose Fill buffer chambers with Tris Glycine buffer Run 45 mintes at 200Volts Stain gel the same way as the previous SDS-PAGE [see page 23]