A detailed explanation of how to how to carry out a chemiluminescent western blot and why each step is taken. Created by a graduate Biochemistry student who is now studying Biological Sciences at Cambridge University. This is designed to be used for all levels of learning, from GCSE to university. ...
Western Blotting
Chemiluminescent Detection Method
Detection of Specific Proteins
DENATURE
1 After lysing and quantifying the protein samples using a
Bradford Assay, the next step is denaturation. This is achieved
by adding a loading buffer, such as Lithium Dodecyl Sulfate
(LDS), along with a reducing agent like ß-mercaptoethanol.
These break any remaining disfulfide bonds and maintain the
protein in a denatured state. The LDS also ensures that the
proteins acquire a uniform negative charge, which is crucial
for their subsequent separation based on size during
electrophoresis.
To further ensure complete denaturation, the samples are
boiled at 80ºC for 10 minutes, which disrupt any tertiary or
quaternary structures, ensuring the proteins are fully
denatured.
GEL ELECTROPHORESIS
2 Next, set up the gel electrophoresis system using Bis-Tris 4-12% Pre-Cast gels. Fill
the electrophoresis tank with MOPS buffer, which helps maintain a stable pH and
keeps the gel cool during the run. Carefully load the protein samples into their
respective lanes, then apply an electric current of 180V for 1 hour. This current
causes the proteins to migrate through the gel matrix towards the anode (positive
end), with smaller proteins travelling faster and thus moving further down the gel.
Bis-Tris 4-12% gels are often preferred over
standard SDS-PAGE gels because their gradient
separates the proteins better over a range of sizes.
Additionally, these gels are less toxic, making them
safer and more convenient to use.
TRANSFER
3 After electrophoresis, the separated proteins
are transferred from the gel onto a
membrane, typically nitrocellulose or PVDF. If
using PVDF, it’s important to soak the membrane
in methanol or transfer buffer to activate it
(makes the membrane porous). Assemble the
transfer “sandwich”, ensuring that each
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