protein separation based on charge and molecular weight

1. KUVEMPU UNIVERSITY
SAHYADRI SCIENCE COLLAGE
DEPARTMENT OF BIOTECHNOLOGY
A Seminar Topic on
“2-D GEL ELECTROPHORESIS”
Submitted by
YOGESHWAR.T
1st MSc Biotechnology
Under the guidance of
Dr. PRADEEP.K
Assistant professor
Department of Biotechnology
Sahyadri science collage
Shivamogga

2. CONTENTS
1.Introduction
2.Gel-Electrophoresis
3.Principle
4.Isoelectric point
5.Isoelectric focusing
6.SDS-PAGE
7.Blotting
8.Conclution
9.References

3. INTRODUCTION
 Two-dimensional gel electrophoresis (2-DE) was first independently introduced by O’Farrell and Klose in
1975.
. It is able to separate hundreds to thousands of proteins or polypeptides by coupling Isoelectric Focusing
(IEF) in first dimension and Sodium Dodecyl Sulphate Polyacrylamide-Gel Electrophoresis (SDS-PAGE) in
second dimension.
: IEF separates proteins in function of their isoelectric point (pI) and SDS-PAGE in function of their
molecular mass

4. Gel-electrophoresis
Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or proteins according to
molecular size. In gel electrophoresis, the molecules to be separated are pushed by an electrical field through a
gel that contains small pores. The molecules travel through the pores in the gel at a speed that is inversely
related to their lengths. This means that a small DNA molecule will travel a greater distance through the gel than
will a larger DNA molecule.
As previously mentioned, gel electrophoresis involves an electrical field; in particular, this field is applied
such that one end of the gel has a positive charge and the other end has a negative charge. Because DNA and
RNA are negatively charged molecules, they will be pulled toward the positively charged end of the gel. Proteins,
however, are not negatively charged; thus, when researchers want to separate proteins using gel electrophoresis,
they must first mix the proteins with a detergent called sodium dodecyl sulphate [SDS]. This treatment makes the
proteins unfold into a linear shape and coats them with a negative charge, which allows them to migrate toward
the positive end of the gel and be separated. Finally, after the DNA, RNA, or protein molecules have been
separated using gel electrophoresis, bands representing molecules of different sizes can be detected.

5. Gel electrophoresis apparatus – an agarose gel is
placed in this buffer-filled box and an electrical
current is applied via the power supply to the rear.
The negative terminal is at the far end (black
wire), so proteins migrates toward the positively
charged anode (red wire).

6. Principle
The principle applied was very simple: proteins were resolved on a gel using isoelectric focusing (IEF), which separates
proteins in the first dimension according to their isoelectric point, followed by electrophoresis in a second dimension in the
presence of sodium dodecyl sulphate (SDS), which separates proteins according to their molecular mass.

7. Isoelectric point
 The isoelectric point (pI, pH(I), IEP), is the pH at which a molecule carries no net electrical charge or is
electrically neutral.
The net charge on the molecule is affected by pH of its surrounding environment and can become more
positively or negatively charged due to the gain or loss, respectively, of protons (H+).
 Biological amphoteric molecules such as proteins contain both acidic and basic functional groups.
 Amino acids that make up proteins may be positive, negative, neutral, or polar in nature, and together give a
protein its overall charge.
 At a pH below their pI, proteins carry a net positive charge; above their pI they carry a net negative charge.
Proteins can, thus, be separated by net charge in a polyacrylamide gel .
A constant pH to separate proteins or isoelectric focusing, which uses a pH gradient to separate proteins.
Isoelectric focusing is also the first step in 2-D gel polyacrylamide gel electrophoresis.
When the environment is at a pH value equal to the protein's pI, the net charge is zero.

9. Isoelectric Focusing(IEF)
This is 1st technique to separate proteins in 2d gel electrophoresis, based on the PI. IEF is performed in 0.5 mm-thin
immobilized pH gradient (IPG)-gel-strips cast on plastic backing. The film-supported gels are easy to handle; IPGs are very
reproducible, in particular because the fixed gradients are not modified by the sample composition; moreover, detergents and
reducing agents can be added without pH gradient disturbing. Samples are usually dissolved in denaturing buffer
IPG strip: A: remove protective film, B: Apply rehydration solution to the strip, C: wet
entire length of IPG strip in rehydration solution by placing IPG strip in strip holder (gel
facing down), D: gently lay entire IPG strip in the strip holder, placing the end of IPG
strip over cathode electrode. E: protein sample can be applied at sample application well
following the rehydration step if the protein sample was not included in the rehydration
solution, F: place cover on strip holder
The protein will run toward their opposite charges to get neutral, the protein will stay
where the PI= 0.(pH=7)
Like this all the proteins get separated based on their charges

10. SDS-PAGE(sodium dodecyl sulphate-poly acrylamide gel electrophoresis)

11. Preparation of polyacrylamide gel
Step-by-step procedure
Gather combs, glass plates, spacer
(silicone tubing), and binder clips.
A comb is used to make wells (lanes) to
load samples. Use an appropriate comb
depending on the sample size.
Example: Use an 8-lane comb for 7
samples and molecular weight markers.
Thoroughly clean the glass plates with
ethanol, and assemble the gel casting
mold.

12. Pour acrylamide solution for a separating gel.
Overlay with water to prevent contact with air
(oxygen), which inhibits polymerization. Allow
acrylamide to polymerize for 20-30 minutes to form
a gel. Remove the overlaid water.
Proteins migrate at different rate depending on the
concentration of the separating gel. Use an
appropriate gel concentration for your target
protein.
Using a higher acrylamide concentration produces
a gel with a smaller mesh size suitable for the
separation of small proteins. In general, an
acrylamide concentration between 6 and 15% is
used.
Gels with an acrylamide concentration gradient
(gradient gels) are also used.

13. Pour acrylamide solution for a stacking gel; insert a
comb and allow the acrylamide to polymerize.
Proteins are highly concentrated when they migrate
through a stacking gel prior to entering a
separating gel. The concentration occurs due to the
difference in the rate of migration of glycine ion,
chloride ion, and proteins, as illustrated below.

14. Preparation of samples
Step-by-step procedure
Add sample buffer to samples, and mix
by flicking the tube.
Heat the samples at 100°C for 3 minutes
in a heat block.
Centrifuge at 15,000 rpm for 1 minute at
4°C, and use the supernatant for SDS-
PAGE.

15. Electrophoresis
Step-by-step procedure
Remove the binder clips, spacer, and
comb from the gel assembly, and mount
the gel in the electrophoresis apparatus
using binder clips.
Pour running buffer into the upper and
lower chambers of the electrophoresis
apparatus, and remove air bubbles and
small pieces of gel from the wells and
under the gel using a syringe.
Load samples and molecular weight
markers in wells.

16. Turn on the power supply, and run the
gel until the dye (BPB) in the sample
buffer reaches the bottom of the gel.
Remove the gel assembly from the
electrophoresis apparatus. Remove the
gel from the glass plates using a spatula,
and prepare for subsequent analysis.

17. Blotting

18. Sandwich model

19. Conclusion
 This is a powerful and widely used method for the analysis of complex protein mixtures extracted from
cells, tissues, or other biological samples. It is the method available which is capable of simultaneously
separating thousands of proteins
 This is a classic and commonly used method for urinary proteome analysis. However, 2-DE is suitable for
large proteins, low molecular weight, and highly hydrophobic proteins. Furthermore, many proteins detected
by this method
 2D-GE was used for protein fingerprinting of Indian black gram varieties.
 Unique and common proteins were identified from these varieties.

20. References
• O'Farrell, PH (1975). "High resolution two-dimensional electrophoresis of proteins". J. Biol. Chem. 250 (10):
4007–21.
• Klose, J (1975). "Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel
approach to testing for induced point mutations in mammals". Humangenetik. 26 (3): 231–43.
• Switzer RC 3rd, Merril CR, Shifrin S (1979). "A highly sensitive silver stain for detecting proteins and peptides in
polyacrylamide gels". Analytical Biochemistry. 98 (1): 231–37.
• Mikkelsen, Susan; Cortón, Eduardo (2004). Bioanalytical Chemistry. John Wiley & Sons, Inc. p.
• Arora PS, Yamagiwa H, Srivastava A, Bolander ME, Sarkar G (2005). "Comparative evaluation of two two-
dimensional gel electrophoresis image analysis software applications using synovial fluids from patients with joint
disease". J Orthop Sci. 10 (2): 160–66. 6.
• Pedreschi R, Hertog ML, Carpentier SC, et al. (April 2008). "Treatment of missing values for multivariate
statistical analysis of gel-based proteomics data". Proteomics. 8 (7): 1371–83.
• Berth M, Moser FM, Kolbe M, Bernhardt J (October 2007). "The state of the art in the analysis of two-
dimensional gel electrophoresis images". Appl. Microbiol. Biotechnol. 76 (6): 1223