Western Blotting

Identification of a specific protein in a complex mixture of proteins can be accomplished by a technique known as Western blotting, named for its similarity to Southern blotting, which detects DNA fragments, and Northern blotting which detects mRNA.
In Western, a protein mixture is electrophoretically separated on an SDS-polyacrylamide gel (SDS-PAGE), a slab gel infused with sodium dodecyl sulfate (SDS), a dissociating agent.
The protein bands are transferred to a nitrocellulose membrane by electrophoresis, and the individual protein bands identified by flooding the membrane with radiolabeled or enzyme-protein of interest.
The Ag-Ab complexes that form on the band containing the protein recognized by the antibody can was bound by a radioactive antibody, its position on the blot can be determined by exposing the membrane to a sheet of x-ray film, a procedure called autoradiography.
However, the linked antibodies against the protein.
After binding of the enzyme antibody conjugate, addition of a chromogenic substrate that produces a highly colored and insoluble product causes the appearance of a colored band at the site of the target antigen.

Even greater sensitivity can be achieved if a chemiluminescent compound with suitable enhancing agents is used to produce light at the antigen site.
Western blotting can also identify a specific antibody in a mixture.
In this case, known antigens of well defined molecular weight are separated by SDS-PAGE and blotted onto nitrocellulose.
The separated bands of known antigens are then probed with the sample suspected of containing antibody specific for one or more of these antigens.
Reaction of an antibody with a band is detected by using either radiolabeled or enzyme-linked secondary antibody that is specific for the spiciest of the antibodies in the test sample.
The most widely used application of this procedure is in confirmatory testing for HIV, where western blotting is used to determine whether the patient has antibodies that react with one or more viral proteins.

Western blotting is primarily used to detect:
a) DNA
b) RNA
c) Proteins
d) Lipids
✔️ Answer: c) Proteins
Western blotting technique is named in analogy with:
a) Eastern blotting
b) Southern blotting
c) Northern blotting
d) Both b and c
✔️ Answer: d) Both b and c
Which technique is used in Western blotting to separate proteins by size?
a) Agarose gel electrophoresis
b) SDS-PAGE
c) Isoelectric focusing
d) Centrifugation
✔️ Answer: b) SDS-PAGE
Which chemical is used to denature proteins during SDS-PAGE?
a) Urea
b) EDTA
c) Sodium dodecyl sulfate (SDS)
d) Formalin
✔️ Answer: c) Sodium dodecyl sulfate (SDS)
After electrophoresis, proteins are transferred to:
a) PVDF paper
b) Nitrocellulose membrane
c) Glass slide
d) Agar plate
✔️ Answer: b) Nitrocellulose membrane

Western blotting detects proteins using specific:
a) DNA probes
b) RNA templates
c) Antibodies
d) Sugars
✔️ Answer: c) Antibodies
Autoradiography is used in Western blotting when the antibody is labeled with:
a) Enzyme
b) Fluorescent dye
c) Radioisotope
d) Alkaline solution
✔️ Answer: c) Radioisotope
A commonly used enzyme in enzyme-linked detection in Western blotting is:
a) Restriction endonuclease
b) Horseradish peroxidase (HRP)
c) RNA polymerase
d) DNA ligase
✔️ Answer: b) Horseradish peroxidase (HRP)
Western blotting is used as a confirmatory test for which viral infection?
a) Hepatitis A
b) HIV
c) Influenza
d) Dengue
✔️ Answer: b) HIV
What type of substrate is used to detect enzyme activity in Western blot?
a) Radioactive
b) Chromogenic or chemiluminescent
c) Lipophilic
d) Alkaline
✔️ Answer: b) Chromogenic or chemiluminescent

In Western blot, proteins are separated based on:
a) Charge
b) Shape
c) Size (molecular weight)
d) Density
✔️ Answer: c) Size (molecular weight)
Which step comes after protein transfer to membrane in Western blotting?
a) Electrophoresis
b) Blocking
c) Precipitation
d) Autoradiography
✔️ Answer: b) Blocking
The blocking step in Western blotting prevents:
a) Protein aggregation
b) Antibody binding
c) Non-specific binding
d) DNA contamination
✔️ Answer: c) Non-specific binding
A commonly used blocking agent in Western blotting is:
a) Glucose
b) Skim milk or BSA
c) Sodium hydroxide
d) SDS
✔️ Answer: b) Skim milk or BSA
Primary antibody in Western blot binds to:
a) Membrane
b) Protein of interest
c) Secondary antibody
d) DNA
✔️ Answer: b) Protein of interest

The secondary antibody in Western blot is usually conjugated with:
a) DNA
b) Fluorophore
c) Enzyme (e.g., HRP)
d) RNA
✔️ Answer: c) Enzyme (e.g., HRP)
Which of the following is a chemiluminescent substrate?
a) TMB
b) Luminol
c) ONPG
d) X-gal
✔️ Answer: b) Luminol
The final colored or luminescent band in Western blot corresponds to:
a) Any protein
b) Only membrane proteins
c) Target protein bound by antibody
d) DNA contamination
✔️ Answer: c) Target protein bound by antibody
The technique most similar to Western blot for DNA is:
a) Northern blot
b) Southern blot
c) ELISA
d) PCR
✔️ Answer: b) Southern blot
Western blotting can also be used to detect specific:
a) Antibodies in a sample
b) Lipids in membrane
c) mRNA expression
d) Sugars in protein
✔️ Answer: a) Antibodies in a sample

Which of the following steps is not involved in Western blotting?
a) Transfer to membrane
b) Probe with antibody
c) Hybridization with DNA probe
d) Electrophoresis
✔️ Answer: c) Hybridization with DNA probe
What is the full form of SDS-PAGE?
a) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
b) Sulfur dioxide sulfate-polymer gel electrophoresis
c) Sodium divalent sulfate-paint gel electrophoresis
d) Solid detergent separation–protein analysis gel electrophoresis
✔️ Answer: a) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Which of these materials is commonly used as the gel matrix in SDS-PAGE?
a) Agarose
b) Polyvinyl
c) Polyacrylamide
d) Cellulose acetate
✔️ Answer: c) Polyacrylamide
The HIV Western blot detects antibodies against:
a) Host DNA
b) Viral proteins
c) Antiviral drugs
d) Cytokines
✔️ Answer: b) Viral proteins
Western blot result is considered positive when:
a) One band is visible
b) No band appears
c) Specific bands corresponding to HIV proteins appear
d) All proteins are detected
✔️ Answer: c) Specific bands corresponding to HIV proteins appear