GROUP 10

Diana M. Nyakudya R203584x

Bevern Zezai R2011486B
Amazinggrace Dube R1916926E
Kelvin Sachiti R

235 RECOMBINANT DNA

TECHNOLOGY
BLOTTING

 technique for analyzing  biological molecules, such as proteins , DNA,

and RNA , involving their separation by gel electrophoresis, transfer to
a nitrocellulose sheet, and subsequent analysis by autoradiography
Types of blotting

 Western blot
 Southern blot
 Northern blot
Western blot

 It is a an often used technique in research to

separate and identify proteins.
 In this technique, a mixture of proteins is
separated based on molecular weight and
type through gel electrophoresis

 The results are transferred to a membrane

producing a band for each protein
How it works

 The western blot test looks for antibodies

against an infection, not necessarily the
infection.
 Is mostly used when one develops a viral,
fungal or bacterial infection, this triggers
immune response against the antigen.
 The antibodies produced by immune cells are
the ones detected by the western blot test.
Steps taken to do the test

 1. sample preparation
 2. gel electrophoresis
 3. blotting to membrane
 4. blocking
 5. protein detection
 6. imaging
 7. analysis
steps Cont…..

 1 Sample preparation
 Protein samples are obtained from tissues or cells or
other solutions which can be analyzed
 The tissue is broken down by blending or
homogenization to extract sample
 A buffer is added to lyses cells and solubilize the proteins
 Buffer should contain protease inhibitors to prevent
degradation of proteins
 Filtration and centrifugation methods are used to further
prepare the samples
2. Gel electrophoresis

 Gel electrophoresis follows, to identify separate

proteins by molecular weight and length
 A positive electrode attracts negatively charged
proteins and the negatively charged electrode
attracts the positively charged proteins
 The previously prepared sample is loaded into the
available polyacrylamide gel
 It is used because of its large pore size and is
suitable for nucleic acid and larger protein
separation (makes it a suitable gel)
Gel electro..... Cont..
Blotting to membrane

 Proteins are placed onto a blotting paper that

is made from material such as nitrocellulose
or nylon
 The transfer process requires voltage to
transfer the proteins from gel to membrane
 Membrane is placed between the gel and the
positive electrode to ensure migration of
negatively charged proteins from gel to the
membrane
blocking

 This is also known as antibody probing and is

used to block out the residual sites on the
membrane
 It prevents unspecific binding of the antibodies
to the membrane, rather than to the protein of
interest.
 Antibody probing usually uses normal goat
serum ,5% Bovine Serum Albumin diluted in Tris
Buffered Saline Tween, or non-fat milk as a
blocking buffer
Cont..

 The procedure consists of incubation of

membrane in the appropriate blocking buffer
for an hour or longer.
 The primary antibody is washed and
removed, only target protein binds.
 Solid support may absorb protein and keep
biological activity unchanged
 the primary antibody is incubated with
labelled secondary antibody
detection

 After washing the labelled secondary antibody binds

with the primary antibody and forms an antibody
complex
 An enzyme is added to the paper.
 If it causes a change in colour, it means antibodies to a
specific infection have been detected.
 The protein-antibody-antibody complex is detected on
the membrane
 There are many labelling ways to a secondary antibody,
including the use of enzymes, fluorophores, gold-
conjugation, and radioisotopes
imaging

 It is used to detect membranes , stained gels,

or for UV light applications.
 The capturing can be analogue using a film,
or digitally performed with a CCD (charge-
coupled device) camera or scanner capturing
the different kinds of emitted signals.
analysis

 Results are analysed and it is the last stage

 In qualitative application, the presence of a
protein is confirmed and the amount is
approximated by visual inspection and the
size is determined by comparison with a
marker
Uses/application

 Commonly used for qualitative detection of

proteins and post translational modification
(phosphorylation)
 Typically used to confirm a positive HIV
diagnosis (by detecting HIV proteins not the
virus)
 Used to confirm positive ELISA
Northern Blot

 A technique based on the blotting principle for

analysis of specific RNA in a complex mixture
 Modified southern blot technique
 Was developed in 1977 by James Alwine, David
Kemp and George Stark at stanford university
 Specifically for RNA identification
 Named it the northern blot just to raise sense of
humour in labs
Procedure

 1 DNA containing a gene of interest is extracted

from human cells and cut into fragments using
restriction enzymes
 2. the fragments are separated according to size
by gel electrophoresis. Each band consists of
many copies of a particular DNA fragment
(visible after staining)
 3. DNA bands are transferred to a nitrocellulose
filter by blotting. The solution passes thru the gel
and filter to the paper towels
Procedure cont…

 4. this produces nitrocellulose filter with DNA

fragments positioned exactly as on the gel
 5. filter is exposed to radioactive labelled
probe for a specific gene. The probe base pair
(hybridise) with a short sequence present on
the gene
 6. filter is exposed to X-ray film. The fragment
containing a gene of interest is identified by a
band on the developed film
Diagram
Advantages

 Particularly used to determine conditions

under which a specific gene can be expressed
 Only mRNA from protein synthesizing cells
will hybridise to probe
 Blots can be stored for years and still be
reprobed for reuse if necessary
Disadvantages

 Risk of mRNA degradation during

electrophoresis
 High radioactive doses are harmful to workers
and environment
 Use of ethidium bromide, DEPC and UV light
needs special attention and training
 Detection with multiple probes is difficult and
time consuming
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