235 Recombinant DNA

technology
Group 8
Diana
Bevern z.
 Blotting

Western blot technique

 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 electropheresis
• 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 lyse 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 membarane
• 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