1. S.Y.BSc SEMESTER III BOTANY PAPER II
UNIT I: INSTRUMENTATION & TECHNIQUES
CHROMATOGRAPHY
BY Miss. Sonam Shukla
Department of Botany
Satish Pradhan Dnyanasadhana College, Thane (w)

2. Horizontal Gel Electrophoresis

3. Agarose gel electrophoresis is a method to separate DNA, or RNA molecules by size.
This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis).
Shorter molecules move faster and migrate farther than longer ones .

4. At any given PH, exist in a solution as electrically charged species either as a cation (+) or anion(-). Under the influence of an electric field these charged particles will migrate either to cathode or anode, depending on the nature of their net charged

5. Electrophoresis is the movement of molecules by an electric current.
Nucleic acid moves from a negative to a positive pole.

6. + - • DNA is negatively charged.
• When placed in an electrical field, DNA will migrate toward the positive pole (anode). H  O2
• An agarose gel is used to slow the movement of DNA and separate by size. + -
Power

7. Components of an Electrophoresis System
Power supply and chamber, a source of negatively charged particles with a cathode and anode
Buffer, a fluid mixture of water and ions
Agarose gel, a porous material that DNA migrates through
Gel casting materials
DNA ladder, mixture of DNA fragments of known lengths
Loading dye, contains a dense material and allows visualization of DNA migration
DNA Stain, allows visualizations of DNA fragments after electrophoresis

8. Electrophoresis Equipment
Power supply
Cover
Gel tank
Electrical leads 
Casting tray
Gel combs

9. Agarose Gel A porous material derived from red seaweed
Acts as a sieve for separating DNA fragments; smaller fragments travel faster than large fragments
Concentration affects DNA migration
Low conc. = larger pores better resolution of larger DNA fragments
1% agarose
2% agarose
High conc. = smaller pores better resolution of smaller DNA fragments

10. Agarose
Buffer Solution
Combine the agarose powder and buffer solution. Use a flask that is several times larger than the volume of buffer.

11. Melting the Agarose Agarose is insoluble at room temperature (left).
The agarose solution is boiled until clear (right).

12. Pouring the gel

13. DNA
buffer  
wells   
Anode
(positive)
Cathode
(negative)

14. Sample Preparation

15. Loading the Gel

16. Running the Gel

17. Electrophoresis Buffer
TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA) are the most common buffers for duplex DNA
Establish pH and provide ions to support conductivity
Concentration affects DNA migration
Use of water will produce no migraton
High buffer conc. could melt the agarose gel
New Sodium Borate (SB) buffer allows gels to be run at higher voltages in less time than traditional buffers
A buffer is a chemical system that maintains a relatively constant pH even when strong acids or bases are added. Buffer solutions contain either a weak acid or weak base and one of their salts. Because a change in pH can alter the charge on a particle, it is important to use a buffer solution when separating during electrophoresis.

18. Loading Dye
DNA samples are loaded into a gel AFTER the tank has been filled with buffer, covering the gel
Contains a dense substance, such as glycerol, to allow the sample to "fall" into the sample wells
Contains one or two tracking dyes, which migrate in the gel and allow monitoring of how far the electrophoresis has proceeded.

19. Allows DNA visualization after gel electrophoresis
DNA Staining
Allows DNA visualization after gel electrophoresis
Ethidium Bromide
In gel staining

20. Analysis
After electrophoresis the gel is illuminated with an ultraviolet lamp to view the DNA bands. The ethidium bromide fluoresces reddish-orange in the presence of DNA.
photograph it with a digital camera.

23. Applications
Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. in restriction mapping of cloned DNA.
Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting
Separation of DNA fragments for extraction and purification.
Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to Northern transfer.

24. VERTICAL GEL ELECTROPHORESIS

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

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

27. 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!

28. 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.

29. Protein Electrophoresis
Separate proteins based on
Size (Molecular Weight - MW)
Allows us to
characterize
quantify
determine purity of sample
compare proteins from different sources

30. 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

31. 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

32. 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.

33. 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.

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

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

36. 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

37. Side view

38. Movement of Proteins in Gel

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

40. 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

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

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

43. 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

44. 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.]

46. 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).

47. 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

48. 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

49. 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

50. 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.
(16 picograms = 16 × grams) in order to be detected. The gel has been stained with Coomassie brilliant blue.

51. Protein bands observed in SDS-PAGE Coomassie Brilliant Blue
Molecular Weight Standard
250 KD
150
100 75 50 37 25 20 15 10

52. Example of silver stained gel
Silver staining is usually times more sensitive than Coomassie Blue staining, but it is more complicated.
Faint but still visible bands on this gel contain less than 0.5 ng of protein!