1. SDS GEL ELECTROPHORESIS
AND BLOTTING TECHNIQUES.
By
Y.PRATHAP
M.Pharm I- Sem (PHARMACEUTICS).
Roll no: 256213886031.
Under the guidance
of:
Asst.Prof . Mr. Uttam
Prasad Panigrahi .
M.PHARM,(PAT).

2. CONTENTS:
SDS Gel electrophoresis.
 Introduction.
 Principle.
 Instrumentation.
 Process.
 Applications.
Blotting techniques.
 Types & applications.
 References.

3. INTRODUCTION OF SDS-PAGE (POLYACRYLAMIDE
GEL ELECTROPHORESIS)
SDS-PAGE, sodium dodecyl sulfate
polyacrylamide gel electrophoresis, is a
technique widely used in biochemistry,
forensics, genetics and molecular biology:
 to separate proteins according to their
electrophoretic mobility (a function of length of
polypeptide chain or molecular weight).
 to separate proteins according to their size, and
no other physical feature.

4. …SDS-PAGE
SDS (sodium dodecyl sulfate) is a detergent
(soap) that can dissolve hydrophobic molecules
but also has a negative charge (sulfATE)
attached to it.

5. Fig.1Before SDS: Protein (pink line) incubated with the denaturing detergent SDS showing
negative and positive charges due to the charged R-groups in the protein.
The large H's represent hydrophobic domains where nonpolar R-groups have collected in an
attempt to get away from the polar water that surrounds the protein.
After SDS: SDS disrupt hydrophobic areas (H's) and coat proteins with many negative
charges which overwhelms any positive charges the protein had due to positively charged R-groups.
The resulting protein has been denatured by SDS (reduced to its primary structure-amino
acid sequence) and as a result has been linearized.

6. ..SDS
 SDS (the detergent soap) breaks up hydrophobic
areas and coats proteins with negative charges
thus overwhelming positive charges in the
protein.
 The detergent binds to hydrophobic regions in a
constant ratio of about 1.4 g of SDS per gram of
protein.
• Therefore, if a cell is incubated with SDS, the
membranes will be dissolved, all the proteins
will be solubalized by the detergent and all the
proteins will be covered with many negative
charges.

7. PAGE
 If the proteins are denatured and put into an
electric field (only), they will all move
towards the positive pole at the same rate,
with no separation by size.
 However, if the proteins are put into an
environment that will allow different sized
proteins to move at different rates.
 The environment is polyacrylamide.
 the entire process is called polyacrylamide
gel electrophoresis (PAGE).

8. ..PAGE
 Small molecules move through the polyacrylamide
gel faster than big molecules.
 Big molecules stays near the well.

9. PRINCIPLE:
 PAGE (Polyacrylamide Gel Electrophoresis), is an
analytical method used to separate components of a
protein mixture based on their size. The technique is
based upon the principle that a charged molecule will
migrate in an electric field towards an electrode with
opposite sign.
 The proteins being covered by SDS are negatively
charged and when loaded onto a gel and placed in an
electric field, it will migrate towards the anode
(positively charged electrode) are separated by a
molecular sieving effect based on size. After the
visualization by a staining (protein-specific) technique.

10. INSTRUMENTATION

11. APPARATUS:
 Apparatus for gel electrophoresis are relatively simple .
Electrophoresis cells are essentially plastic boxes with anode and
cathode compartments. Electrodes(usually platinum wire) and jacks
for making electrical contact with the electrodes.
 Gels are held vertically between the electrode chambers during the
run. Gel cassettes have open tops and bottoms. The bottom is sealed
with a gasket during gel formation and the top is open to receive
monomer solution. The top and bottom ends are open and in contact
with buffer for electrophoresis.
 High voltage direct current supplies provide electrical power for
electrophoresis.
 Micropipettes, test tubes and heating blocks are the sample handling
necessities.

12. PARTS OF THE SYSTEM
 Gel support medium
 Agarose.
 Polyacrylamide(PA).
 Detergent: sodium dodecyl sulfate(SDS).
 Buffer : the electrical current in an electrophoresis cell is
carried largely by the ions supplied by the buffer compounds.
Proteins constitute only a small portion of the current carrying
ions in an electrophoresis cell. So buffers supply current
carrying ions, maintain desired PH, provide a medium for heat
for dissipation . Ex : Tris-acetate-EDTA and Tris- borate -
EDTA.
 DC Power supply.

15. PROCESS:
Sample preparation.
Prepare 2X sample buffer consisting of 0.5MTris-HCl,
pH 6.8, 4.4% SDS, 300mMMercaptoethanol,
10mg/ml Bromophenol Blue and mix with equal volume
of sample .Bring to 95° C for 10 minutes, cool to room
temperature before loading. If particulate is present,
centrifuge samples 5 minutes at 14k RPM in
microcentrifuge, and load the gel.

16. ENTIRE PROCESS DIAGRAM OF SDS GEL
ELECTROPHORESIS.

17. PROTEIN VISUALIZATION ON GELS
• Immediately after electrophoresis proteins in the gels are
precipitated by either adding alcohol containing
solutions or strong acids (e.g. TCA)
• DNA may be visualized using ethidium bromide.
 Protein are often stained by Coomassie Brilliant Blue
dye or by photography-like treatment with AgNO3
(silver staining)
 There are many other stains available (e.g. Stains-all,
fluorescence probes etc.)

18. PROTEIN GEL (SDS-PAGE) THAT HAS BEEN
STAINED WITH COOMASSIE BLUE.

19. EXAMPLE OF SILVER STAINED GEL

20. APPLICATIONS:
 SDS PAGE is a useful method for separating and
characterizing macromolecules like DNA, RNA and
proteins.
 In Forensic , DNA Fingerprinting: men proving or
disproving paternity by this technique. Used as witness.
 The human Genome project.
 Illness: It can help scientists to identify certain damaged
genes . It can also help to identify certain genetic
diseases like sickle cell anemia, also identify viruses.
 Blotting : Separation of restricted genomic DNA prior to
southern blotting and RNA prior to northern blotting.

21. BLOTTING
TECHNIQUES.

22. WHAT IS BLOTTING?
Technique for transferring
DNA
RNA
 Proteins
onto a carrier so they can be separated,
and often follows the use of a gel
electrophoresis.

23. TYPES OF BLOTTING TECHNIQUES
BLOTTING
TECHNIQUES
Southern Blot
It is used to detect
the DNA.
Northern blot
It is used to detect
the RNA.
Western blot
It is used to detect
proteins.

24. BLOTTING SHEET
Whatman 3mm paper….. world’s most widely used
blotting paper.
 REASON????
 high quality
 purity
 consistency

25. CREATING THE SANDWICH
 The sandwich consists of :
filter paper
Nitrocellulose membrane
gel matrix
another piece of filter paper

26. SOUTHERN BLOTTING
History:
 Sir Edwin Southern
 Developed in 1975

27. SOUTHERN BLOTTING
“Used to detect the DNA”
 This method Involves:
 Separation
 Transfer
 Hybridization.
 This DNA can be:
 Single gene
 Part of a larger piece of DNA……..viral genome
“The key to this method is Hybridization”

28. HYBRIDIZATION
“Process of forming a
dsDNA molecule between a
ssDNA probe and a ss-target
patient DNA”

29. PRINCIPLE
 The mixture of molecules is separated.
 Immobilized on a matrix.
 Probe addition to the matrix to bind to the molecules.
 Unbound probes are removed.
“The place where the probe is connected
corresponds to the location of the immobilized
target molecule.”

30. STEPS IN SOUTHERN BLOTTING
The DNA is digested
Fragments
Gel electrophoresis
Transfer to membrane
Probing
Autoradiogram

32. APPLICATIONS
Southern blotting is used in:
 Gene discovery
Mapping
 Evolution
 Development studies
 Diagnostics
 Forensics

33. NORTHERN BLOTTING
History:
Northern blotting was developed by James
Alwine and George Stark at Stanford University.
Northern blotting is a technique for
detection of specific RNA sequences

34. STEPS INVOLVED IN N.B
RNA isolation
Loading of sample on Agarose gel
Blotting on nitrocellulose membrane
Labeling with probe
Washing to remove unbound probe
Detection by autoradiogram

36. APPLICATIONS
 A standard for the direct study of gene expression at the
level of mRNA (mRNA transcripts)
 Detection of mRNA transcript size
 Study RNA degradation
 Study RNA splicing - can detect alternatively spliced
transcripts
 Study RNA half-life
 Study IRES (internal ribosomal entry site) – to remove
possibility of RNA digestion vs. 2nd cistron translation.

37. WESTERN BLOTTING
Discovery???
 Dr. Douglas Lake of the University of Arizona School of
Medicine's Department of Microbiology and Immunology
“A technique in which proteins are
separated by gel electrophoresis and
transferred to a membrane sheet. A
specific protein is then identified through
its reaction with a labeled antibody.”

38. PRINCIPLE
This technique works on
the principle on
“Antigen-Antibody”
relationship

39. PREREQUISITE FORW.B
•The SDS
PAGE technique is a
prerequisite for Western
blotting.
“SDS (sodium dodecyl sulfate) is a
detergent (soap) that can dissolve
hydrophobic molecules but also has
a negative charge (sulfate) attached
to it.”

40. STEPS INW.B
1. Gel electrophoresis:
The proteins are separated according to size.
2. Membrane Transfer:
Transferring to nitrocellulose by applying
current.
3. Blocking:
Done to prevent non-specific protein interactions
between the membrane and the antibody protein.

42. APPLICATIONS
 To identify the specific proteins
 To identify their masses
 The confirmatory HIV test to detect anti-HIV antibody in a human
serum sample.
 The definitive test for Bovine spongiform encephalopathy (BSE,
commonly referred to as 'mad cow disease').
 Some forms of Lyme disease testing also employ Western blotting.

43. REFERENCES:
 Introduction to Biotechnology by W.J. Thieman and
M.A. Palladino. Pearson & Benjamin Cummings
2nd edition.
 http://www.toodoc.com/SDS-PAGE-ppt.html
 http://www.bio.davidson.edu/courses/genomics/met
hod/Westernblot.html
 http://en.wikipedia.org/wiki/Dot_blot
 Bio analytical techniques by M.L.Srivastava
 http://amrita.vlab.co.in/index.php.
Wikipedia.
 www.authorstream.com.