Preparation, Purification and Identification

of Lysozyme (Part Three)

• This part continues with the gel separation of
lysozyme. Click to review the part one and part
two.
 1.2 Gel separation of lysozyme

• Gel filtration, also known as molecular sieve filtration, or exclusion

chromatography. Its outstanding advantage is that the gels used in
the chromatography are inert carriers, having no charge, weak
adsorption force, mild operating conditions, and can be carried out
over a wide temperature range. In the meantime, organic solvents
are not needed. It has a good separation effect for the polymer
material. Gel is a three-dimensional network structure and porous
insoluble bead-like particulate material. It is used to separate
substances, mainly based on the porous gel with different radius of
the protein molecules (nearly spherical) with different exclusion
effect achieved. That is, it is based on the molecular size of the
physical properties of the separation and purification.
• For some types of gels, some macromolecules can not enter the
interior of the gel particles and are completely excluded from the
column. They can only flow out of the column along the gaps
between the particles. Some small molecules can not diffuse and
permeate freely into the gel inside the sieve, and then taken out of
the eluate. The smaller the molecule, the deeper it enters the gel
and the more it travels. Therefore, small molecules eventually flow
out of the column, whereas the macromolecules flow out of the
column first. Some medium-sized molecules are between
macromolecules and small molecules, and can only enter part of
larger pores of the gel, that is, they are partially expelled. Therefore,
the order in which these molecules flow out of the column is also
between large and small molecules . After the sample was gel-
chromatographed, the molecules flow out in descending order to
achieve the purpose of separation.
• 20.Adjust the pH to 5.5 with 1mol/L NaOH, centrifuge, discard the
sediment and collect the purified liquid;

• 21. Put the purified solution into the dialysis bag, dehydration
concentration with research fine polyethylene glycol;

• 22. Take a certain amount of Sephadex G-50 in 250mL beaker and

add 120mL eluent, swelling at room temperature for 2-3 days,
pouring repeatedly to remove fine particles, and then remove air
from the gel gap;

• 23. Take a clean glass column and vertically fix on the iron platform,
at a distance of about 3cm from the upper end of the column, close
the column outlet, add deionized water, open the outlet until the
liquid level down to the column mark;
• 24. Add the eluate (about 1/3 bed height) in the column, slowly pour
the gel concentrated slurry into the column; open the outlet until the
gel deposition is about 1-2cm height. Then close the outlet, let it
stand for a moment, wait for the gel to completely settle, connect the
constant flow pump, and balance the column with 1-2 times column
bed volume eluant to make the column bed stable;

• 25.Aspirate the eluate from the top of the column (be careful not to
disturb the surface), open the outlet, the residual liquid down to the
plastic tangent (enough to not glue), close the outlet, with a fine tube
to draw 0.5mL (2mg/mL) blue dextran-2000;
• 26.Add the lysozyme sample solution (0.5-1mL) according to the
operation method of 22, elute and collect the eluate at a rate of 1.5
mL / tube for 5 min;

• 27. Set the elution conditions with chromatography system and elute.
Record the absorbance of the eluent at A280 recorded by the
recorder, and directly plot the elution curve. The eluate from the
collection tube in the elution peak was pooled and stored at -20 ° C
for later use.
 1.3 SDS polyacrylamide gel identification of
isolated products
• 28.Rapidly fill the gap between the two glass plates with the
acrylamide solution to leave room for perfusion of the concentrated
gel (comb teeth plus 0.5). Carefully inject a layer of water over the
glue surface to prevent oxygen from entering the gel solution;

• 29.After the separation gel polymerization is complete (about 30min),

pour out cover the water layer, and then use filter paper to wash
residual water;

• 30.Prepare a concentrated gel, pour the concentrated gel directly

onto the polymerized gel, Immediately insert a clean comb in the
concentrate solution and place the gel vertically at room temperature;
• 31.While waiting for concentrated gel polymerization, the lysozyme
sample can be processed; add sample processing solution at a
volume ratio of 1: 1, heated at 100 ℃ for 5min and then put on ice,
12000rpm centrifugal 3min, take 10uL supernatant point kind;

• 32.After the concentrated gel polymerization is complete, carefully

remove the comb, fix gel plate on the electrophoresis device, the
upper and lower trough each added Tris-Gly electrode buffer;

• 33.Connect the electrophoresis device to the power supply, adjust

the voltage to 200V, electrophoresis until bromophenol blue reaches
about 1cm above the bottom of the separation gel, turn off the power;
• 34.remove the glass plate from the electrophoresis device, pry open
the glass plate with a spatula, and cut corners for marking if
necessary;

• 35.Stain the glue with a liquid dye for 1-2h;

• 36.Decolorize the gel with decolorizing liquid for 1h, if necessary,

replace the decolorization solution until the background is clear;

• 37.After decolorization, the gel can be immersed in water to save, or

take a photo of the gel, or dry the gel rub the film;

• 39. Measure and calculate lysozyme molecular weight.

 • Thanks for watching!
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