proteincharacterization/...

Protein Characterization/Purification

Dr. Kevin Ahern

Protein Purification• Applications of Biochemistry Knowledge


• Opening Cells


• Opening Cells• Centrifugation


• Opening Cells• Centrifugation• Fractionation

Dialysis• Applications of Biochemistry Knowledge

Dialysis• Applications of Biochemistry Knowledge

• Separates salts from proteins

Chromatography (Column)• Applications of Biochemistry Knowledge


• Separation based on charge - Ion Exchange


• Separation based on charge - Ion Exchange • Separation based on size - Size Exclusion / Gel Filtration


• Separation based on charge - Ion Exchange • Separation based on size - Size Exclusion / Gel Filtration• Separation based on affinity - Affinity Chromatography


• Separation based on charge - Ion Exchange • Separation based on size - Size Exclusion / Gel Filtration• Separation based on affinity - Affinity Chromatography• Separation based on polarity - Reverse Phase Chromatography

Ion Exchange Chromatography

Cation exchange chromatography (+ sticks) Anion exchange chromatography (- sticks)

Cation Exchange 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 materials The most polar materials will elute first. The most non-polar materials will elute last.

Agarose Gel Electrophoresis


Mesh-like support


Mesh-like supportEvenly charged rod-like molecules (negative)


Mesh-like supportEvenly charged rod-like molecules (negative)Samples loaded in wells


Mesh-like supportEvenly charged rod-like molecules (negative)Samples loaded in wellsElectrical current pushes molecules through support


Mesh-like supportEvenly charged rod-like molecules (negative)Samples loaded in wellsElectrical current pushes molecules through supportAll molecules have same mass to charge ratio


Mesh-like supportEvenly charged rod-like molecules (negative)Samples loaded in wellsElectrical current pushes molecules through supportAll molecules have same mass to charge ratioLargest molecules move slowest


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


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



Largest

Loading Wells



Largest

Smallest

Loading Wells

Polyacrylamide Gel Electrophoresis


Mesh-like support - tinier pores


Mesh-like support - tinier poresNegatively charged rod-like molecules (SDS-protein)


Mesh-like support - tinier poresNegatively charged rod-like molecules (SDS-protein)Samples loaded in wells


Mesh-like support - tinier poresNegatively charged rod-like molecules (SDS-protein)Samples loaded in wellsElectrical current pushes molecules through support


Mesh-like support - tinier poresNegatively charged rod-like molecules (SDS-protein)Samples loaded in wellsElectrical current pushes molecules through supportAll molecules have same mass to charge ratio


Mesh-like support - tinier poresNegatively charged rod-like molecules (SDS-protein)Samples loaded in wellsElectrical current pushes molecules through supportAll molecules have same mass to charge ratioLargest molecules move slowest



Largest



Largest

Smallest

Isoelectric Focusing

Metabolic Melodies

The Proteins Marching One by One To the tune of "The Ants Go Marching One by One”

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


In Proteomics, Researchers Aim to Quantitate All of the Proteins Made in Cell/Tissue2-D Gel Electrophoresis is One Way to Do This Analysis

Add Protein Mixture to Polyelectrolyte Column


Apply Electrical Current


Apply Electrical Current

Proteins Separate According to pI Values

High pI

Low pI


Rotate Apply to

Gel


Add SDS

Separate By Size on

Polyacrylamide Gel

Rotate Apply to

Gel


Add SDS

Separate By Size on

Polyacrylamide Gel

Separated By Charge/pI

Rotate Apply to

Gel


2-‐D Gel Electrophoresis


Separated By Size



Separated By Size Each Spot Corresponds to a Unique Protein



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



Label Proteins Blue



Label Proteins Blue

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



Label Proteins Blue



Blue - Proteins in a Cancer Cell,

But Not a Healthy Cell

Black - Proteins Equally Abundant in Both Cells

Microarray Analysis Allows a Researcher to Measure the Quantity of every mRNA of Interest Made in a Cell/Tissue

Microarray Analysis

Chemically Synthesize DNA Corresponding to an mRNA

Microarray Analysis

Chemically Synthesize DNA Corresponding to an mRNA Bond Thousands of Copies of That DNA to a Spot on a Slide

Microarray Analysis

Repeat for Every mRNA of an Organism

Microarray Analysis

Repeat for Every mRNA of an Organism One Spot Per mRNA

Microarray Analysis

Gene #1

Microarray Analysis

Gene #1

Gene #2

Microarray Analysis

Gene #1

Gene #2Gene #3

Microarray Analysis

Take Two Sets of Cells - Healthy vs Cancerous

Microarray Analysis


Isolate All mRNAs from Each

Microarray Analysis


Copy Each mRNA Using Reverse Transcriptase

to Make cDNA Copies of Each

Microarray Analysis


Add Fluorescent Green Tag to Normal Cell cDNAs

Microarray Analysis


Add Fluorescent Green Tag to Normal Cell cDNAs

Add Fluorescent Red Tag to Cancer Cell mRNAs

Microarray Analysis

Microarray Analysis

Mix cDNA Samples

Microarray Analysis

Mix cDNA Samples

Pour Mixture Onto Slide

Microarray Analysis

Mix cDNA Samples

Pour Mixture Onto SlideAllow Hybridization to Occur

Microarray Analysis

Mix cDNA Samples

Pour Mixture Onto SlideAllow Hybridization to Occur

Wash Unhybridized Samples Away

Microarray Analysis

Intensity of Color Measures Amount of mRNA

Microarray Analysis

Intensity of Color Measures Amount of mRNAShade of Color Measures Relative Expression Between Cell Types

Microarray Analysis


Bright Green - Abundant In Healthy Cells, Not in Cancer Cells

Microarray Analysis


Bright Green - Abundant In Healthy Cells, Not in Cancer Cells Bright Red - Abundant In

Cancer Cells, Not in Healthy Cells

Microarray Analysis




Bright Yellow - Abundant In Both Cells Types

Microarray Analysis




Bright Yellow - Abundant In Both Cells Types

Black - Absent in Both Cells Types

Microarray Analysis

Microarray Analysis

Western Blotting

Useful for identifying proteins in a gel

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

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

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

Detection uses Labeled Antibody Specific to Protein of Interest

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

Metabolic Melodies

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

by Kevin Ahern and Indira Rajagopal

I’ve just run a gel. I do not think it went too well

I may have used a bit much SDS. The stacker’s looking like a mess.

It’s true Oh now what will I do?

The protein sample’s my last one.

To purify it was not fun I spent three weekends working late.

The middle lanes aren’t looking great. I’m screwed

Good God what will I do?

Crawling. I’m almost bawling

The boss is calling to follow through

I just loaded all I’ve got to make this final western blot

My fingers are both crossed for sure I hope my protein product’s pure.

I do Then my thesis is through

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

by Kevin Ahern and Indira Rajagopal

I’ve just run a gel. I do not think it went too well

I may have used a bit much SDS. The stacker’s looking like a mess.

It’s true Oh now what will I do?

The protein sample’s my last one.

To purify it was not fun I spent three weekends working late.

The middle lanes aren’t looking great. I’m screwed

Good God what will I do?

Crawling. I’m almost bawling

The boss is calling to follow through

I just loaded all I’ve got to make this final western blot

My fingers are both crossed for sure I hope my protein product’s pure.

I do Then my thesis is through

Hating. All of the waiting

I’m contemplating what I should do

Staining. My eyes are straining

There’s no complaining. I say ‘wahoo’

‘Cuz it has the band I need I’ll go and have it scanned to speed

The writing of my thesis and Proceed onto the post-doct’ral plan

Oh that will be so grand

Pieces make up my thesis. No more ‘phoresis. The promised land.

Writing so unexciting.

But no more biting. My nails again.

Writing is coinciding. With reference citing. I’m at the end.

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