Ion exchange laboratory
Download
1 / 31

Ion Exchange Laboratory - PowerPoint PPT Presentation


  • 213 Views
  • Uploaded on

Ion Exchange Laboratory. Pre-Lab Discussion Outline. Column chromatography Types and principles Focus on: ion exchange chromatography Ion exchange experiment Sample mixture: GFP and cytochrome c Pour column Purification protocol Spetrophotometer For your information slides

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Ion Exchange Laboratory' - santiago


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

Pre lab discussion outline
Pre-Lab Discussion Outline

Column chromatography

Types and principles

Focus on: ion exchange chromatography

Ion exchange experiment

Sample mixture: GFP and cytochrome c

Pour column

Purification protocol

Spetrophotometer

For your information slides

Spectrophotometer

Standard curve


Types of column chromatography
Types of Column Chromatography

Ion Exchange

Gel Filtration

Affinity


Basis for separation
Basis for separation

Ion Exchange

Gel Filtration

Affinity

Charge

Size

Conformation


Ion exchange chromatography
Ion Exchange Chromatography

Separates molecules based on charge

A solid charged cellulose matrix with a charge is used

Solution of different charges are used


Types of ion exchangers
Types of Ion Exchangers

A cation exchanger

An anion exchanger


Today s ion exchange experiment
Today’s Ion Exchange Experiment

Objective: to separate two proteins of different charges from a sample mixture using an ANION EXCHANGER and then determine the concentration of one of these proteins using spectrophotometry.


Example of a biomedical application
Example of a biomedical application

  • Let’s say you have a population of cells (tumor + non-tumor) and the tumor cells express an abnormal protein of a different charge (but the same size) as the normal protein. You think this abnormal protein may contribute to tumor growth.

  • You want to separate these proteins and study them.


Obtain sample mixture of two proteins of different charges
Obtain sample mixture of two proteins of different charges

Green fluorescent protein(GFP): negatively charged (anionic) chromophores (26.9 kDa)

395 nm (major peak)

(NOTE: due to simulated dye product we will use 550 nm)!

Cytochrome c: positively (cationic) charge protein

(12.4 kD)


GFP

238 amino acids

Used to follow gene expression


Cytochrome c
Cytochrome c

Highly conserved heme-protein

Associated with inner mitochondrial membrane

Participates in electron transport




Overview of your ion exchange experiment
Overview of your ion exchange experiment:

Separate GFP from cytochrome c

Add 0.01 M KOAc first

First remove (elute) and discard cytochrome c

Then add 0.5M KOAc next

Remove (elute) and save GFP


Add 0 5 ml sample mixture 1 mg ml of starting gfp to anion exchanger column
Add (0.5 ml) sample mixture (1 mg/ml of starting GFP) to anion exchanger column

Sample mixture:

cytochrome c positively charged (cationic)

and

GFP negatively charged (anionic)

Beads have a positive charge


0.01 M Potassium Acetate

Cytochrome c elutes but GFP remains bound to column

How do you remove GFP from the bead?


Now add the:

0.5 M Potassium acetate

GFP now elutes

Collect sample

Measure volume

Determine concentration


Measure the concentration of gfp by spectrophotometry
Measure the concentration of GFPby spectrophotometry


1 st calibration
1st Calibration

Empty

Zero transmission

Pure water

100% transmittance


Next gfp standard curve using serial dilutions
Next: GFP standard curve using serial dilutions:

* Stock GFP is 1 mg/ml

BE SURE YOU UNDERSTAND HOW TO PREPARE SERIAL DILUTIONS


Finally measure the gfp collected from the column
Finally measure the GFP collected from the column

  • Obtain absorbance values for your GFP collected from your column

  • Use your standard curve to convert absorbance to concentration

  • Sample mixture contained 1 mg/ml of GFP. (remember you added 0.5ml of this solution to your column)


Ion exchange worksheet
Ion exchange worksheet :

Please hand in a neatly presented:

1. Table with absorbance and corresponding concentration values

2. A graph of your GFP standard curve, be sure to label the axis

3. The concentration of your purified GFP(mg/ml)

4. The total amount of GFP purified (mg)

5. The volume (ml) of your collected (purified) GFP

6. The % recovery

= mg GFP purified/total amount GFP (mg) addded x 100

Note: If you diluted your purified GFP remember to use the dilution factor in your calculations


Let’s Begin….

These additional slides are to help you review the principles of spectrophotometry and the use of a standard curve.


Instrumentation review
Instrumentation: Review

Spectrophotometer

T

A

0

100

plug


Calibration
Calibration

Spectrophotometer

Cuvette

(requires 4 ml)

T

A

0

100

plug

Set: 100% transmission with cuvette + water

Set: 0% transmission without cuvette


What is a standard curve
What is a standard curve?

  • A graph that allows a quantitative determination known concentration.


Why do we use standard curves
Why do we use standard curves?

To obtain quantitative measurements

In clinical settings

Measurement of blood hormones

Measurement of environmental carcinogens

Measurement of drugs

Measurement of antibodies (such as anti-HIV)


Example of standard curve step 1 data collection of known values
Example of Standard CurveStep 1: Data Collection of Known Values

Absorbance 260 nm

DNA ug/ml

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.18

0.35

0.60

0.70

0.95


Step 2 standard curve
Step 2. Standard Curve

1.0

.8

.6

.4

Absorbance (260nm)

(dependent variable)

.2

0

.2

.4

.6

.8

1.0

0

DNA (ug/ml)

(independent variable)


Step 3 use the curve to calculate unknowns
Step 3. Use the curve to calculate unknowns

1.0

.8

Unknowns

Prostate Tumor DNA Abs. 0.85

Normal Prostate DNA Abs. 0.40

.6

.4

Absorbance (280nm)

(dependent variable)

.2

= unknown values

X

X

0

X

.2

.4

.6

.8

1.0

0

DNA (ug/ml)

(independent variable)


Fyi ion exchange chromatography ph
FYI: Ion Exchange Chromatography & pH

Generally speaking, a protein will bind to a cation exchange resin if the buffer pH is lower than the isoelectric point (pI) of the protein, and will bind to an anion exchange resin if the pH is higher than the pI.


ad