1. Sodium DodecylSulphate- PolyAcrylamide Gel Electrophoresis (SDS-PAGE)
Exercise 10

2. Experimental Goals To understand the principle of SDS-PAGE
To become familiar with the SDS-PAGE setup

3. What is Electrophoresis?
Electrophoresis is a laboratory technique for separating molecules based on their charge

4. Separation of a Mixture of Charged Molecules
Charged molecules are separated based on their electrical charge and size within a matrix
Positive Molecules
Analyze
Identify
Purify
Size Separation
Charge Separation
Mixture of Charged Molecules
Negative Molecules

5. The gel (matrix)
The gel (matrix) itself is composed of either agarose or polyacrylamide.
Polyacrylamide is a cross-linked polymer of acrylamide.
Acrylamide is a potent neurotoxin and should be handled with care!

6. Polyacrylamide gels
Have smaller pores than agarose, therefore high degree of resolving power.
Can separate DNA fragments which range in size from bp.
DNA fragments which differ in size by one nucleotide can be separated from each other.
Polyacrylamide gel electrophoresis is also used to separate protein molecules.

7. Protein Electrophoresis
Separate proteins based on
Size (Molecular Weight - MW)
Allows us to
characterize
quantify
determine purity of sample
compare proteins from different sources
And it is a step in Western blot

8. Protein Electrophoresis
Proteins, unlike DNA, do not have a constant size to charge ratio
In an electric field, some will move to the positive and some to the negative pole, and some will not move because they are neutral
Native proteins may be put into gel systems and electrophoresed
An alternative to native protein gels forces all proteins to acquire the same size to charge ratio

9. SDS-PAGE
SDS-PAGE ( sodium dodecylsulphate-polyacrylamide gel electrophoresis)
The purpose of this method is to separate proteins according to their size, and no other physical feature
In order to understand how this works, we have to understand the two halves of the name: SDS and PAGE

10. Sodium Dodecylsulphate
Since we are trying to separate many different protein molecules of a variety of shapes and sizes,
we first want to get them to be linear
no longer have any secondary, tertiary or quaternary structure (i.e. we want them to have the same linear shape).
Not only the mass but also the shape of an object will determine how well it can move through and environment.
So we need a way to convert all proteins to the same shape - we use SDS.

11. Sodium Dodecylsulphate
SDS (sodium dodecyl sulfate) is a detergent that can dissolve hydrophobic molecules but also has a negative charge (sulfate) attached to it.
If SDS is added to proteins, they will be soluablized by the detergent, plus all the proteins will be covered with many negative charges.

12. Sodium Dodecylsulphate
A sample of protein, often freshly isolated and unpurified, is boiled in the detergent sodium dodecyl sulfate and beta-mercaptoethanol
The mercaptoethanol reduces disulfide bonds
The detergent disrupts secondary and tertiary structure
The end result has two important features:
all proteins contain only primary structure and
all proteins have a large negative charge which means they will all migrate towards the positive pole when placed in an electric field.
They migrate through a gel towards the positive pole at a rate proportional to their linear size
Molecular weights with respect to size markers may then be determined
The detergent binds to hydrophobic regions in a constant ratio of about 1.4 g of SDS per gram of protein, (every 3 aa)

13. Sodium Dodecylsulphate
Now we are ready to focus on the second half - PAGE.

14. SDS and Proteins
SDS
Protein

15. SDS and Proteins
SDS nonpolar chains arrange themselves on proteins and destroy secondary tertiary and quarternary structrure
So much SDS binds to proteins that the negative charge on the SDS drowns out any net charge on protein side chains
In the presence of SDS all proteins have uniform shape and charge per unit length

16. Polyacrylamide Gel Electrophoresis (PAGE)
PAGE is the preferred method for separation of proteins
Gel prepared immediately before use by polymerization of acrylamide and N,N'-methylene bis acrylamide.
Porosity controlled by proportions of the two components.
autopolymerisation of acrylamide takes place. It is a slow spontaneous process by which acrylamide molecules join together by head on tail fashion A solution of these polymer chains becomes viscous but does not form a gel, because the chains simply slide over one another. Gel formation requires hooking various chains together.

17. Catalyst of polymerization
Polymerization of acrylamide is initiated by the addition of ammonium persulphate and the base N,N,N’,N’-tetrametyhlenediamine (TEMED)
TEMED catalyses the decomposition of the persulphate ion to give a free radical

18. Polymerization of acrylamide

19. Polymerization of acrylamide
Cross-linked polyacrylamide gels are formed from the polymerisation of acrylamide monomer in the presence of smaller amounts of N,N’-methylenebisacrylamide (bis-acrylamide)
Bisacrylamide is the most frequently used cross linking agent for polyacrylamide gels
Temed

20. Polyacrylamide Gels
Bis-Acrylamide polymerizes along with acrylamide forming cross-links between acrylamide chains

21. Polyacrylamide Gels
Pore size in gels can be varied by varying the ratio of acrylamide to bis-acrylamide
Protein separations typically use a 29:1 or 37.5:1 acrylamide to bis ratio
Lots of bis-acrylamide
Little bis-acrylamide

22. Side view

23. Movement of Proteins in Gel

24. Movement of Proteins in Gel
smaller proteins will move through the gel faster while larger proteins move at a slower pace

25. Components of the System
DC Power Source, Reservoir/Tank, Glass Plates, Spacers, and Combs
Support medium
Gel (Polyacrylamide)
Buffer System
High Buffer Capacity
Molecules to be separated
Proteins
Nucleic Acids

26. Vertical Gel Format: Polyacrylamide Gel Electrophoresis
Reservoir/Tank
Power Supply
Glass Plates, Spacers, and Combs

27. Step by Step Instructions on how to assemble the polyacrylamide gel
apparatus

28. Procedure Prepare polyacrylamide gels
Add diluted samples to the sample buffer
Heat to 95C for 4 minutes
Load the samples onto polyacrylamide gel
Run at 200 volts for minutes
Stain

29. Gel Preparation Reagent 8% (Running Gel) 5% (Stacking Gel)
Acrylamide/ Bisacrylamide (40%) *
4.0 mls
2.5 mls
1 M Tris-HCl pH 8.8
7.5 mls
water (distilled)
8.2 mls
9.7 mls
10% SDS
200 µl
10% Ammonium Persulfate
100 µl
TEMED (added last)
10 µl
* = 19:1 w:w ratio of acrylamide to N,N'-methylene bis-acrylamide
The purpose of the stacking gel is to condense the protein into a tight band before running it through the separating gel. This allows better resolution in the separating gel. The stacking effect works because the stacking gel is made up of a lower concentration of polyacrylamide than the separating gel. The protein stacks because the proteins move very quickly through the stacking gel, only to run into the separating gel. The proteins at the top of the stacking gel then have time to catch up with the proteins at the bottom.
[The other relevant difference between the stacking gel and the separating gel is pH, which affects the charge and thus the migration of glycine, a component of the electrophoresis running buffer. In the stacking gel (at pH 6.8), glycine "trails" the proteins and facilitates their stacking. At the separating gel pH of 8.8, glycine is negatively charged and thus runs at the dye front, leaving the proteins to separate out behind it.]

31. Gel Preparation
Mix ingredients GENTLY! in the order shown above, ensuring no air bubbles form.
Pour into glass plate assembly CAREFULLY.
Overlay gel with isopropanol to ensure a flat surface and to exclude air.
Wash off isopropanol with water after gel has set (+15 min).

32. Sample Buffer Tris buffer to provide appropriate pH
SDS (sodium dodecyl sulphate) detergent to dissolve proteins and give them a negative charge
Glycerol to make samples sink into wells
Bromophenol Blue dye to visualize samples
Heat to 95C for 4 minutes

33. Loading Samples & Running the gel
Run at 200 volts for minutes
Running Buffer, pH 8.3 Tris Base       12.0 g Glycine          57.6 g SDS                4.0 g
distilled water to 4 liter

34. SDS-PAGE
SDS-PAGE
Denature protein(s) 100ºC, heat in presence of 1% SDS and 0.1M MCE
This gives proteins uniform shape and constant q/m
SDS binds denature protein 1.4g SDS/g Protein primarily through hydrophobic interactions with its long C12 tail, the SO4 head group decorates the protien with negative charges to give proteins constant q/m
Thus, in an SDS-PAGE experiment electrophoretic mobility depends primarily on size, m. wt., since q/m and shape are essentially equal.
? Small molecules have a greater mobility than large molecules due to?
A- sieving effect of the gel matrix
? Because
A- all proteins under the same acceleration by E.
Thus SDS-page is used to separate proteins by their molecular weight, identify proteins by their molecular weight or estimate an unknown protein’s molecular weight. The latter is done by plotting log MWT for some standard proteins of known molecular weight, run at the same time in the same gel , versus their relative mobility (*distance traveled into the gel relative to a dye front for instance). This technique works for proteins and protein subunits 10,000 – 200,000 mwt.
Apparatus- vertical, vertical minis, tube and horizontal or flatbed design
Two electrodes [anode (+) and cathode (-)] in conductive buffer:
2e- + 2H2O → 2OH- + H2↑ H2O → 2H+ + ½ O2↑ + 2e-
HA + OH- → A- + H2O H+ + A- → HA
Apply equal and constant voltage on all cross-sectional areas of solid support. Electric field (volts/cm)
Ohm’s law (E = IR) voltage is a function of current (I) and resistance (R) and since electrophoresis apparatus and buffers give a constant resistence, current is often used to define the voltage requirements
?What carries the current?
A- Proteins and Nuclein Acids and all ions in solution
Therefore we perform GE experiments in low ionic strenth buffer 0.05 – 0.15 M so thecurrent is not swamped with small molecule ions such that the proteins experimnce very little of the current and at pH 9 where most porteins have a negative charge and will migrate toward the anode

35. Staining Proteins in Gels
Chemical stains detect proteins based on differential binding of the stain by the protein molecules and the gel matrix.
They are nonspecific in action, detecting proteins without regard to their individual identities.
The important characteristics for a useful stain are: low background, high sensitivity, large linear range and ease of use.
(16 picograms = 16 × grams) in order to be detected. The gel has been stained with Coomassie brilliant blue.

36. Staining Proteins in Gels
Coomassie Brilliant Blue
The CBB staining can detect about 1 µg of protein in a normal band.
Silver Staining
The silver stain system are about 100 times more sensitive, detecting about 10 ng of the protein.
How to Quantify Proteins ?
(16 picograms = 16 × grams) in order to be detected. The gel has been stained with Coomassie brilliant blue.
Densitometry

37. Molecular weight estimation by SDS-PAGE
Molecular Weight Standard
250 KD
150
100 75 50 37 25 20 15 10

38. Molecular weight estimation by SDS-PAGE
Calibration curve for molecular weight estimation.

39. Western Blotting