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Western Blot
Western Blot
Ganpat University
(
Northern blot for RNA
Western blot
for Proteins
Proteins.
BY,
Nirmal Patel
Southern blot for DNA
DNA.
M Pharm (Bio Tech)
The Western blot is an analytical
technique used to detect specific
proteins in a given sample of
tissue homogenate or extract.
-može da se koristi za dokazivanje At ili Ag
The name Western blot was given to the technique by W. Neal Burnette.
WB
It uses gel electrophoresis to separate
native or denatured proteins
by the 3-D structure of the protein by the length of the polypeptide
(native conditions) (denaturing conditions)
Steps in a Western blot:
Contents:
- Tissue preparation
- Gel electrophoresis
- Transfer
- Blocking
1. Gel za koncentrovanje
-blago kiseo (pH 6.5)
-niska koncentracija AA (5%) → porozan
2. Gel za separaciju
- bazan (pH 8.8)
- viša koncent. AA (6-18%) → kanali (pore) su uži
(pore)
-They Are traveling to the + since they have – charge due to SDS.
----------------
----------------
-These strands go throught the tunnel and are seperated by
their
size!!!
size !!! tj.
molekulske težine
Denatured
Proteins
Proteins here are actually linearized.
Boja:
-Bromphenol blue
-Pyronin
- The entire stack is placed in a buffer solution which moves up the paper by
capillary action, bringing the proteins with it. it
- Two step:
step: 1. 2.
Detection -Biotin ili
+ supstrat
-Enzim (AP, HRP)
- One step
Historically, the probing process was performed in two steps
because of the relative ease of producing primary and
secondary antibodies in separate processes.
one-step probing systems that would allow the process
one-
to occur faster and with less consumables.
consumables
• Detekcija Ag
U smeši proteina dobijenih liziranjem μ.o. (k.ć. sa
virusom, bakterije?), identifikuju se Ag upotrebom At
obeleženih enzimima.
Npr. Dg HIV infekcije analizom seruma na At:
Medical diagnostic applications:
- definitive test for
Bovine Spongiform Encephalopathy (BSE).
-Western blot can also be used as a confirmatory test for Hepatitis B infection.
Advantages
Specific interaction of antibody and antigen can
be directly visualized.
Disadvantages
Technically demanding
Expensive
Subject to interpretation
Presence or absence of bands
Intensity of those bands
When should WB be used?
WB assay should not be used as a - screening test.
HOWEVER: