protein purification slides

Warm up! Sit next to your lab partner from yesterday Put on a lab coat and gloves. Take out your lab notebook.

Title: Bacterial Transformation Purpose: To change the phenotype (physical traits) of e.coli by

changing the geneotype (DNA or genes) Method: See ScienceBridge Protocol Results: “Wait for Quanina to explain”

Take a look at your results. Did you see what you expected?

Results Number of fluorescent colonies counted:

If you saw colonies growing that were not fluorescent, explain how this could have happened?

If you saw no colonies growing, how could this have happened?

If your results were unexpected, explain how this could have happened.

Conclusion

Protein PurificationStudent Training

Objective:

To extract and separate fluorescent proteins in E.coli from other cellular debris (membrane, other proteins, DNA, RNA, etc.)

Transformation

Bacteria now express cloned fluorescent protein…

Bacterial chromosome

Allow bacteria to grow for 1-3 days on plate with ampicillin.

Plasmid Uptake of foreign DNA, often a circular plasmid

Bacterial chromosome

Warm-up Sit next to your lab partner from yesterday. Take out your PD and reading packet annotations. Swap

PDs and annotate. Repeat until 3 people have read and annotated your PD. Do this without talking

SLCs Yay!Take out a sheet of paper.

Write down 3 things you are proud that you have accomplished this semester.

Write down 2 things you want to improve on this semester.You can talk about any class you are taking this

semester or you can talk about the semester overall.

Purify a specific protein from over 4,000 naturally occurring E. coli gene products.

How do you purify proteins?

Organisms produce thousands of different proteins, each having a different function…

HormonesHair and Nails

Structural Support

Enzymes

Muscle Contraction

Antibodies

Receptors, membrane channels

Nutrient Storage

Proteins……are created by living organisms

(DNA → RNA → PROTEIN → trait)

…have unique structures that determine function(insulin, cobratoxin, fluorescence)

…can be isolated from living things(humans, cobras, jellies)

…can be studied and modified by humans(fluorescent proteins)

Protein Structure

Protein Structure1° = amino acids

2° = basic structure(hydrogen bonds)

3° = 3D structure

4° = interactionof subunits

Why Purify Proteins?

Research

• to make vaccines • to treat disorders

• to understand structure

Medicine

Pancreas → Identify Cells → Isolate Gene → Insert Gene

→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein

→ Human Use

Why Purify Proteins?

Insulin for diabetics

Pancreas → Identify Cells → Isolate Gene → Insert Gene

→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein

→ Human Use

Why Purify Proteins?

Insulin for diabetics

Pancreas → Identify Cells → Isolate Gene → Insert Gene

→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein

→ Human Use

Why Purify Proteins?

Insulin for diabetics

Pancreas → Identify Cells → Isolate Gene → Insert Gene

→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein

→ Human Use

Why Purify Proteins?

Insulin for diabetics

Pancreas → Identify Cells → Isolate Gene → Insert Gene

→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein

→ Human Use

Why Purify Proteins?

Insulin for diabetics

Pancreas → Identify Cells → Isolate Gene → Insert Gene

→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein

→ Human Use

Why Purify Proteins?

Insulin for diabetics

Pancreas → Identify Cells → Isolate Gene → Insert Gene

→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein

→ Human Use

Why Purify Proteins?

Insulin for diabetics

Pancreas → Identify Cells → Isolate Gene → Insert Gene

→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein

→ Human Use

Why Purify Proteins?

Insulin for diabetics

How do you purify proteins?

Fluorescent proteins are just one of thousands of

proteins in the cell!

Fluorescent Protein

Purify a specific protein from over 4,000 naturally occurring E. coli gene products.

How do you purify proteins?

How do we purify proteins? (3)

1. Break open the cells in 2 ways

- Adding Lysozyme

- Snap Freeze

0. Scrape cells into the tube with TE Buffer

Lysozyme is a naturally occurring enzymethat is used to break open cells.

Lysozyme

How do we purify proteins? (3)2. Centrifuge the cells to separate the heavy cell material from lighter cell material supernatant

pellet

How do we purify proteins? (3)3. Separate the fluorescent protein from other light cellular debris using column chromatography

3a. Mix supernatant with nickel beads

3b. Pass the supernatant and nickel bead mixture through the column and into a waste tube

3c. Add elution buffer to column and collect solution in a new tube

How do the nickel beads work?

his-his-his-h

is-his-h

is

His- tag: a chain of histidine amino acids

How do the nickel beads work?

Ni2+

Ni2+

Ni2+

Ni2+Ni2+

Ni2+

Ni2+

The his-tag on the fluorescent protein and nickel bind like 2 magnets

How does the elution buffer work?

Elution buffer contains a molecule called imidizole that has a stronger attraction to the nickel beads

Ni2+

Ni2+

Ni2+

Ni2+

Ni2+

Ni2+

How do we purify proteins?

Finished! Now you have a pure sample containing only fluorescent proteins

Why purify proteins?In Research: In Medicine:

To characterize protein structure, function and interactions

To create vaccines from recombinant proteins such as insulin and factor 8

Tricky Parts of Lab Scraping cells into tube

Using the correct buffer solution