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Protein Electrophoresis:

SDS-PAGE
(Sodium dodecyl sulfate- polyacrylamide gel electrophoresis)

Alex de Lencastre
Bio 606
9/26/2013
Learning Objectives

• Explain the difference between native and denaturing


electrophoresis.

• Describe the roles of SDS and Beta-mercaptoethanol in SDS-PAGE.

• Understand how to choose the correct percentage of acrylamide


and the correct protein ladder in our gels.

• Understand how a stacking gel works and why we use it.

• Understand how to prepare samples for SDS-PAGE, load and run


an SDS-PAGE gel and stain the gel to determine if your Ni-affinity
chromatography purification worked
Review – What have we done so far?

Express 6-His-GBP in bacteria.

1. Break cells – obtain soluble fraction.


2. Ni-NTA Affinity Purification.
3. Concentration Determination (Bradford Assay).

This week:
4. Estimation of purity and expression of our 6-His-GBP
protein using SDS-PAGE.
Next week: REVIEW
Following week: EXAM - I & Notebook Grading - I
PAGE
PAGE = Polyacrylamide Gel Electrophoresis

Electrophoresis: subjecting charged molecules to an electric field

Polyacrylamide: The gel material – a crosslinked-slab of “mesh” material that


slows or retards the movement of macromolecules.
PAGE – native or denaturing

Native PAGE: molecules are separated in their “native” or fully-


folded state.

Under “non-denaturing”
or “native” conditions,
rate of movement in gel
depends on size, shape
and charge.

COMPLICATED!
PAGE – native or denaturing

Denaturing PAGE: molecules are denatured before


electrophoresis and made uniformly negative.

Migration becomes proportional to


protein size = Much easier to interpret.
Role of BME and SDS

Beta-Mercaptoethanol (BME) is a reducing


agent that breaks di-sulfide bonds.

Sodium dodecyl sulfate (SDS) is a detergent


that binds to proteins, forcing them denature.

SDS is also a salt, with negative charge.


It coats the protein and makes the protein
negatively charged.

The amount of SDS bound is proportional to the size of the protein.


As a consequence, the charge of the protein-SDS complex is
proportional to its length.
(Poly)- Acrylamide Gels (PAGE)
What is PAGE Gel made of?
PAGE Gel: A mixture of acrylamide and bis-acrylamide, polymerized
and crosslinked
Bis-acrylamide necessary for
crosslinking. The extent of crosslinking
determines the MW range of separation.

Crosslinking creates a polymer with a


network of branches – a “mesh” that
slows down the movement of large
molecules.
The % of acrylamide determines the
number of crosslinks – higher
percentage = more mesh = slower
movement of molecules.

When would we use a high percent vs low percent gel?


(Poly)- Acrylamide Gels

8% 10%

Resolution power at different acrylamide %:

- A lower % acrylamide gel to resolve bigger


macromolecules.

- A High % acrylamide gel to resolve smaller


macromolecules.
(Poly)- Acrylamide Gels – Stacking/Resolving

“Discontinous PAGE”

2 different gels stacked on top of


each other.

Stacking gel This improves resolution:


Stacking lets all proteins get “lined
up” properly - they arrive at the
running gel at the same time.
Running gel
(Poly)- Acrylamide Gels – Stacking/Resolving
Glycine Stacking gel

Running gel
Stacking Gel: pH (6.8) Running Gel: pH (8.8)
Glycine net charge is Glycine net charge is
barely negative much more negative

Glycine ionization
Net charge
+1 0 -1

At pH ABOVE the pI, glycine is


deprotonated and becomes
more negatively charged.
(Poly)- Acrylamide Gels – Stacking/Resolving
Buffer Systems

-- Tris acts as the cation:

-- Can use various things to acts as the anion:


- Glycine
- Acetate
- Hepes
- MOPS
- MES
Protein Ladders

A set of 10 pre-stained protein bands, with known size, in the range of 6-180kDa

The 6HIS-BGP protein


is ~16kDa

Run your gel at the same time as the protein ladder (standards) and then compare
the size of your protein to the size of the standards.
Loading a gel

Sample (or Loading) Buffer:

Your sample +

- SDS
- BME
- Glycerol
- Dyes

Heat (70oC) - 10 minutes


Staining Polyacrylamide Gels
Coomassie brilliant blue
Fast (10 min – 1 hour)
Detection limit: 50 ng protein per band

Silver stain
Slow (2-3 hours)
Detection limit: 100 times greater than Coomassie
Example SDS-PAGE Gel

Predict:
What do you think gel
will look like?

Consider adding those


predictions to your
notebook.