Protein

Characterization/Purification
Dr. Kevin Ahern

Protein Purification
Applications of Biochemistry Knowledge

Opening Cells
Centrifugation
Fractionation

Dialysis

Applications of Biochemistry Knowledge

Separates salts from proteins

Chromatography (Column)
Applications of Biochemistry Knowledge

Separation
Separation
Separation
Separation

based
based
based
based

on
on
on
on

charge - Ion Exchange

size - Size Exclusion / Gel Filtration
affinity - Affinity Chromatography
polarity - Reverse Phase Chromatography

Ion Exchange Chromatography

Cation exchange chromatography (+ sticks)

Anion exchange chromatography (- sticks)

Cation Exchange Chromatography

Size Exclusion / Gel Filtration Chromatography

Size Exclusion / Gel Filtration Chromatography

Affinity Chromatography

Reverse Phase HPLC Chromatography

Reverse Phase HPLC Chromatography

Columns have non-polar packing material

Non-polar materials interact more with column than polar materia
The most polar materials will elute first.
The most non-polar materials will elute last.

Agarose Gel Electrophoresis

Mesh-like support
Evenly charged rod-like molecules (negative)
Samples loaded in wells
Electrical current pushes molecules through support
All molecules have same mass to charge ratio
Largest molecules move slowest

Agarose Gel Electrophoresis

Mesh-like support
Evenly charged rod-like molecules (negative)
Samples loaded in wells
Electrical current pushes molecules through support
Loading Wells
Largest molecules move slowest
Largest

Smallest

Agarose Gel Electrophoresis

Mesh-like support
Evenly charged rod-like molecules (negative)
Samples loaded in wells
Electrical current pushes molecules through support
Largest molecules move slowest

Polyacrylamide Gel Electrophoresis

Mesh-like support - tinier pores

Negatively charged rod-like molecules (SDS-protein)
Samples loaded in wells
Electrical current pushes molecules through support
All molecules have same mass to charge ratio
Largest molecules move slowest
Largest

Smallest

Isoelectric Focusing

The Proteins Marching One by One

To the tune of "The Ants Go Marching One by One
Metabolic Melodies
Lyrics by Tari Tan

Oh there's a method you should know that's very huge

It's spinning round and round inside the centrifuge
The supernatant, pellet too
You choose the one that's right for you
And from there we pu-ri-fy
What's inside
To size exclude filtration is the way to go
The beads have pores small proteins can go in you know
The largest ones, they come out fast
The smallest ones eluting last
And the proteins purified
By their size
Electrons power gel e-lec-tro-pho-re-sis
The protein is denatured thanks to SDS
Proteins in a minus state
Get sorted by atomic weight
Smaller ones in speedy mode
To the anode
Ion exchange is special chromatography
To switch cations, you must have a minus bead
Upon this bead, the proteins bind
They're positive, not any kind
And the others wash right through
Out to you
Oh my this song has given you a mighty list
Perhaps we'll just skip over ol' dialysis
So study HPL and C
If you have questions, talk to me
You will get through protein hell
You'll do well.

Proteomics - 2D Gel Electrophoresis

In Proteomics, Researchers Aim to Quantitate All of the Proteins Made in Cell/Tissue

2-D Gel Electrophoresis is One Way to Do This Analysis

Proteomics - 2D Gel Electrophoresis

Add Protein Mixture to Polyelectrolyte Column

Proteomics - 2D Gel Electrophoresis

Apply Electrical Current

High pI
Proteins Separate According to pI Values
Low pI

Proteomics - 2D Gel Electrophoresis

Separated By Charge/pI
Rotate
Apply to
Gel
Add SDS

Separate By
Size on
Polyacrylamide
Gel

2-D Gel Electrophoresis

Proteomics - 2D Gel Electrophoresis

Separated By Charge/pI

Separated By Size

Each Spot Corresponds

to a Unique Protein

The Intensity of Each Spot is a Measure of the Amount of Protein Present

2-D Gel Electrophoresis

Biotechnology
Proteomics

Take Two Sets of Cells - Healthy vs Cancerous

Label Proteins
Orange
Orange - Proteins in a
Healthy Cell,
But Not a Cancer Cell

Label Proteins
Blue
Blue - Proteins in a
Cancer Cell,
But Not a Healthy Cell
Black - Proteins Equally
Abundant in
Both Cells

Microarray Analysis

Gene #3
Gene #2
Microarray Analysis Allows a Researcher to Measure the
Quantity of every mRNA of Interest Made in a Cell/Tissue
Gene #1
Chemically Synthesize DNA Corresponding to an mRNA
Bond Thousands of Copies of That DNA to a Spot on a Slide
Repeat for Every mRNA of an Organism
One Spot Per mRNA

Microarray Analysis

Take Two Sets of Cells - Healthy vs Cancerous

solate All mRNAs from Each

Copy Each mRNA Using

Reverse Transcriptase
to Make cDNA Copies of Each

Add Fluorescent Green Tag

to Normal Cell cDNAs

Add Fluorescent Red Tag

to Cancer Cell mRNAs

Microarray Analysis

Mix cDNA Samples

Pour Mixture Onto Slide

Allow Hybridization to Occur
Wash Unhybridized Samples Away

Microarray Analysis

Intensity of Color Measures Amount of mRNA

Shade of Color Measures Relative Expression Between Cell Types

Bright Green - Abundant In

Healthy Cells, Not in Cancer Cells

Black - Absent in
Both Cells Types

Bright Red - Abundant In

Cancer Cells, Not in Healthy Cells

Bright Yellow - Abundant In

Both Cells Types

Microarray Analysis

Western Blotting

Useful for identifying proteins in a gel

https://en.wikipedia.org/wiki/Western_blot#/media/File:SDS-PAGE_sample.png

Western Blotting

Proteins Must be Transferred from Gel to a Membrane

https://en.wikipedia.org/wiki/Western_blot#/media/File:Western_blot_transfer.png

Western Blotting

etection uses Labeled Antibody Specific to Protein of Intere

https://en.wikipedia.org/wiki/Western_blot#/media/File:Western_Blot_binding.png

Metabolic Melodies Ive Just Run a Gel

(to the tune of "I've Just Seen a Face")

by Kevin Ahern and Indira Rajagopal

Ive just run a gel.

I do not think it went too well
I may have used a bit much SDS.
The stackers looking like a mess.
Its true
Oh now what will I do?

Hating.
All of the waiting
Im contemplating what I should do

The protein samples my last one.

To purify it was not fun
I spent three weekends working late.
The middle lanes arent looking great.
Im screwed
Good God what will I do?

Cuz it has the band I need

Ill go and have it scanned to speed
The writing of my thesis and
Proceed onto the post-doctral plan
Oh that will be so grand

Crawling.
Im almost bawling
The boss is calling to follow through

Pieces make up my thesis.

No more phoresis. The promised land.

I just loaded all Ive got

to make this final western blot
My fingers are both crossed for sure
I hope my protein products pure.
I do
Then my thesis is through

Staining.
My eyes are straining
Theres no complaining. I say wahoo

Writing so unexciting.
But no more biting. My nails again.
Writing is coinciding.
With reference citing. Im at the end.