David Bui Richard Lauhead Randall Mello Michelle Tran.

Slides:

Advertisements

Similar presentations

Solutions Containing More Than One Solute SM Buffer TE Buffer NOTE: To successfully accomplish this section you must be familiar and comfortable with all.

Advertisements

Hemoglobin A 2 Practical Hematology Lab - LAB 6 -.

BISC 220 Lab 2 Protein Purification by Affinity Chromatography & Determination of Specific Activity.

Monoprotic Acid-Base Equilibria Monoprotic Weak Acids Monoprotic Weak Bases Fraction of Dissociation-Association Salts of Weak Acids Buffers.

David Bui Randall Mello Richard Lauhead Michelle Tran.

David Bui Richard Lauhead Randall Mello Michelle Tran Team E.

1 Chapter 10 Acids and Bases 10.10Dilutions. 2 Dilution Diluting a solution Is the addition of water. Decreases concentration. ConcentratedDilutedSolution.

Enzyme-linked Immunosorbent Assay

Genetic Incorporation of Unnatural Amino Acids into Proteins Monica Amin 1, Yang Song 2, Yan Liu 2, Harbani Malik 2, Vipul Madahar 2, Jiayu Liao 2 Department.

Updates on Prion Protein Purification Donovan Duggan Bow Suriyamongkol Supervisor: Dr. David Wishart Prion Group Meeting 2 February, 2007.

Solution Concentration. Calculations of Solution Concentration: Mass Percent Mass percent Mass percent is the ratio of mass units of solute to mass units.

One Stage Factor V Assay

Topic: Dilution Do Now:

SPECTROPHOTOMETRY cont.. Application qualitative A Absorbance spectrum.

Buffers AP Chemistry.

Ion Exchange Chromatography. Cation exchangers They contain immobilized anionic groups that bind to cations. e.g. a matrix with carboxymethyl group(CM)

TOPIC: Concentration and Dilution Do Now:. Parts of a Solution SoluteSolute = dissolved substance SolventSolvent = dispersing medium.

THE MATHEMATICS IN A TITRATION CURVE (WITH A LITTLE BASE 10 AND LOGARITHM ARITHMATIC ADDED)

Ion Exchange Laboratory

Buffer Solutions a weak acid and its conjugate base, or

Gel filtration chromatography y/college/voet/ /animat ed_figures/ch05/f5-6.html.

BIOLOGY 3020 Fall 2008 “Keys of Corn” Project Plasmid isolation Genetic Diversity in corn. Lots of different types of corn are offered for sale at the.

Introduction The determination of protein-protein interaction affinity is very important in studying cell signaling pathways such as the SUMO pathway,

Extraction and Quantitation of DNA From E. coli

Ion Exchange Laboratory

ACIDS AND BASES Acid Base Titration A very accurate method to measure concentration. Acid + Base  Salt + Water H + + OH -  H 2 O Moles H + = Moles.

Expression, Purification and Isolation of the MinE protein By Arsalan Wasim and Nicholas Wong.

Enzyme Linked Immunosorbent Assay (ELISA). ELISA Enzyme Linked Immunosorbent Assay (ELISA) Term Was Coined By Engvall and Pearlmann in 1971 Different.

Honors Review Questions 47.4 g of propane is combusted in the lab, 17.4 L of product gas was collected at a temperature of 37.1 o C and 734 Torr. What.

Chapter 14 Acids and Bases. Chapter 14 Table of Contents Copyright © Cengage Learning. All rights reserved The Nature of Acids and Bases 14.2Acid.

Introduction to Analytical Chemistry

Quality Assurance How do you know your results are correct? How confident are you?

Molarity By: Gurpreet Dulay & Kiranjit Kandola Some definitions The CONCENTRATION of a substance in solution is the amount of the substance which exists.

Protein-Protein Binding Kit of SUMO Conjugation Cascade by FRET Technology David Bui Richard Lauhead Randall Mello Michelle Tran.

% by Mass Another way to measure the concentration of a solution % by mass = mass solute x 100 mass solution Solution = solute + solvent.

Acid – Base Equilibria Buffer Solutions: –Question: Was the ICE Problem set up needed? –Answer: No. The assumption of x

Ch. 13/14: Solutions Describing a Solution’s Composition.

The Common Ion & Buffering. Common Ion Effect the ionization of an acid (or base) is limited by the presence of a significant amount of its conjugate.

The effect of salts and chaperone proteins on Alkaline Phosphatase stability Aka: the results of 271 continuous enzyme activity assays Mary Klein Dhruve.

1 Acid-Base Titration and pH Chapter Self-Ionization of water Two water molecules produce a hydronium ion and a hydroxide ion by transfer of a proton.

Making Molar Solutions From Liquids (More accurately, from stock solutions)

Dilution Problems. 2-step Molarity Problems Study them because that’s the process we need to go through if we are making a solution from a solid solute.

Solution Preparation and Dilutions. Solution Preparation How do we create chemical solutions in a laboratory setting with a certain concentration/molarity?

Protein Purification by Ion Exchange Chromatography

Titration. Definition Process used to determine the strength of an unknown acid or base Can determine the unknown acid by adding to it a base of known.

Concentration of Solutions What is Concentration? What is Concentration? The amount of solute dissolved in a specified amount of solution The amount of.

Welcome.  Saturday School: 10-11ish  Monday: Cold Call Quiz.

PREPARING SOLUTIONS AND REAGENTS II. SOLUTIONS WITH MORE THAN ONE SOLUTE RECIPE I Na 2 HPO 4 6 g KH 2 PO 4 3 g 0.4% glycerol 10 mL Dissolve in water.

Solution Chemistry. Lab solutions ………“like a recipe!”  Components required (or materials)  Quantities required (correct volumes and concentrations)

Purification of immunoglubin by ion exchange chromatography Bahiya Osrah

Chapter 15. Acid Any solution with more H 3 O + ions than OH - Electrolytes Taste sour pH less than 7 React with many metals Strong acid-any acid that.

Protein Overexpression in E. coli and

Estimation of quantity and quality of isolated DNA

Hydronium Ions and Hydroxide Ions Self-Ionization of Water In the self-ionization of water, two water molecules produce a hydronium ion and a hydroxide.

One-Stage Quantitative

Practical Hematology Lab

Ion Exchange Chromatography

Solution Concentration

Solution Concentration

NH4+ (aq) H+ (aq) + NH3 (aq)

DNA Extraction and Purification

Experiment 7 Preparation and Properties of Buffers

Solution Concentration

Week 5. Buffers solutions

Ion Exchange Laboratory

Quantitative Proteins Estimation by Lowry method

Unit 3, Lesson 14: Dilutions

DNA precipitation (Mini-prep)

Solution Concentration

Titration of cellulose and resistance to elution conditions.

Presentation transcript:

David Bui Richard Lauhead Randall Mello Michelle Tran

 Goal: Development of FRET based kit to screen compounds that could alter binding between SUMO1 and UBC9. Why is it important to have this kit? Disregulation of the SUMO pathway has been linked to diseases including ovarian carcinoma, melanoma, and lung adenocarcinoma. (Mo and Moschos 2005)

 Bradford Assay:  Total protein concentrations of solution can be calculated using the equation obtained from graph.

 Using highly pure proteins, serial dilutions were done to make solutions at different protein concentrations  Values range from 1 ng to 100 ug

 For accurate fluorescence measurements of single proteins and conjugations, an assay must have values away from background noise  500 ng is the lowest amount for usable assay conditions. Ypet-Ubc9 (ng) RFU without blank Cypet-Sumo1 (ng) RFU without blank

IngredientsConcentration of Solutions(M) Wash1Protocol 1Protocol 2Protocol 3 NaCL Tris HCL pH Wash2 NaCL Tris HCL pH Triton0.50%2.00%1.25% Wash3 NaCL Tris HCL pH Immidazole Elution NaCL0.3 Tris HCL pH Immidazole Resuspension BufferConcentration(M) NaCl0.5 Tris-HCl pH Immidazole0.005 Cell Lysate obtained from 1 Liter of solution and resuspended in 30 mL of Resuspension buffer was obtained. Column purification protocol involves 10mL of lysate poured into a column with 500 uL of agarose nickel bead solution with subsequent 10 mL washes. Elution took place 500 uL at a time and continued until the beads showed no fluorescence.

 Using the Bradford Assay to determine total protein concentration and the fluorescence curves generated from the sensitivity tests, the purity was calculated for each protocol Ypet-UBC9 Purification protocol Bradford concentrations(ng/uL) fluorescent concentration (ng/uL)Purity Protocol Protocol Protocol Cypet-SUMO1 Purification protocol Bradford concentrations(ng/uL) fluorescent concentration (ng/uL)Purity Protocol Protocol Protocol

Ypet-UBC9 could be around 70% pure Cypet-SUMO1 is unlikely to be 97% pure Ypet-UBC9Cypet-SUMO1

Keeping a constant fluorescent protein amount at 1 ug. BL21 cell lysate proteins were added to change percent purity. Results show that purity has little effect on fluorescence at 1ug of fluorescent protein.

Tested purity effects on FRET with each protein at a constant amount of 500 ng. Results demonstrate that purity of fluorescent proteins and in FRET has no effect at low concentration(10ng/ul). Emission max of Ypet-Ubc9 over Cypet-SUMO1 to obtain FRET ratio. Results demonstrate little to no change in FRET ratio when purity is varied.

SUMO ASSAY KIT 1/18/20101/25/2010 2/1/20102/8/2010 2/15/20102/22/2010 3/1/20103/8/2010 3/15/20103/22/20103/29/2010 4/5/2010 4/12/20104/19/20104/26/2010 5/3/2010 5/10/20105/17/20105/24/20105/31/2010 6/7/2010 TasksStartEnd %comple te Purification optimization1/18/20102/8/201090% Flexstation 2 fluorescence sensitivity test1/18/20102/8/ % Dialysis/Lyophilization2/1/20102/22/2010 Protein purity effects2/1/20102/22/201050% Expression optimization3/1/20103/15/2010 Fret sensitivity3/1/20103/15/2010 compound screening sensitivity3/22/20104/5/2010 Stability testing3/22/20104/5/2010 Oxidation testing4/12/20104/26/2010 Kit assembly4/12/20104/26/2010 Add secretion factors to proteins5/3/20105/31/2010