A.
Principle :
Dialysis
is a separation process depends on the differential transport of solutes of
different sizes across a porous barrier separating two liquids where the
driving force is concentration gradient
only. It is usually used to separate solutes too large to diffuse through the
barrier from those small enough to diffuse through it.
B.Material:
a.Spectra
/ Por Dialysis membrane.
b.Closures
for Dialysis membrane
c.0.01 M
Sodium phosphate buffer, pH 7.2
(Appendix - I)
d.Antibody
to be desalted (Human Ig)
e.Magnetic
stirrer and magnetic bar.
C. Method:
1.Cut the
required length of flat dialysis membrane and place in distilled water.
2.Open the
membrane by gently squeezing
from one end to another
and wash the inner side of membrane by passing the distilled
water gently without damaging the membrane.
3.Close
one end of the membrane using closure or thread.
4.Pour the
antibody solution through another end and close the same.
5.Place
the filled bag in a beaker containing 0.01M SPB and magnetic bar and keep the
beaker on a magnetic stirrer and adjust the speed (at 4 0C).
6.Change
the pre-cooled buffer after every 4-6 hr for three time (1 lit each time)
7.Collect
the antibody.
Note :
1.Choice
of the pore size of the membrane depends on the substance to be dialysed.
2.
For critical work such as
enzyme studies etc. extensive cleaning of the membrane is necessary
before use.
3.The
membrane should not dry because it may damage the membrane.
4.It is
advisable to test the system for leaks using distilled water before adding the
protein solution to the tubings.
Ultramembrane Filtration
A.Principle
:
Ultrafiltration sometimes called reverse
osmosis is a process in that the solutes and solvent, up to a certain critical
size are forced through the barrier by high pressure on one side of the porous
barrier than the other. Since there is always a flow of solvent moving along the
solute it can be used for the concentration process.
B.Materials
:
a.Ultrafiltration
tubing.
b.Closures
for the tubing.
c.Conical
flask with side arm.
d.Funnel
and single-holed rubber cork.
e.Sample
to be concentrated.
C.Method :
1.Cut
10-15 cm ultrafiltration tubing and wash it as mentioned in Dialysis.
2.Insert
the tip of the funnel at one end of the
tubing and pass
the end of
funnel with membrane through the
rubber cork.
3.Close
the other end of tube with closure or with thread.
4.Pour the
sample to be concentrated through the funnel.
5.Apply
vacuum pressure through the side arm and close it. Leave the flask at 4 0C.
6.Collect
the sample by opening the tied end and dialyse if necessary.