Western blot protocol using nitrocellulose membrane

Western blot protocol

An example of protocol and comparison result for HanoBlot
nitrocellulose membrane:

Protocol:

1. Three gels were cast.
2. The same protein sample was loaded at 2, 4, and 8 micro-liters
into separate lanes as a set.
3. After electrophoresis, the gels were sliced and each set of
protein samples was overlaid on top of the membranes. Proteins
on gels were transferred onto the membranes.
4. After the transfer, proteins on the membranes were stained with
dye (Amido Black)
5. The membranes were incubated in 5% skimmed milk overnight
to block the extra protein binding space.
6. Antibodies to the proteins were added, further incubated for 3
hours.
7. The membranes were washed with buffer containing detergent
(0.05% Tween 20) 5 times up to an hour.
8. Secondary antibodies conjugated with enzyme (peroxidase)
were added to the membranes and further incubated for 3 hours.
9. Extensively washed again and finally developed with enzyme
substrate.

The results on the right showed that HanoBlot NC membrane is
better or equivalent to the competitor’s product.