2. Page 2
This part is to give an explanation about the first step—Preparation of
lysozyme. And the next post gonna talk about the gel separation of lysozyme.
3. Page 3
Experimental steps
1.Experimental methods and steps:
This experiment mainly utilized 724 acid cation exchange resin adsorption,
(NH4) 2SO4 salting-out, ph adjustment isoelectric point removal of hybrid
protein, gel layer sucked separation and purification, and finally the purified
product was identified by SDS-PAGE.
4. Page 4
1.1 Preparation of lysozyme
Lysozyme was prepared with egg white as raw material and
lysozyme was extracted by resin adsorption method. Because
lysozyme is a basic protein, it is adsorbed by the cation exchange
resin in a near-neutral environment. Using this property, it can be
separated from other proteins in the egg, and then purified by (NH4)
2SO4 salting-out and pH adjustment isoelectric point to remove the
impurity protein. The obtained lysozyme can be crystallized.
5. Page 5
1.Soak the resin with 1mol / L HCl for 2h, and wash with deionized
water to near neutral;
2.Soak the resin with 1mol / L NaOH for 2h, and wash with deionized
water to near neutral;
3.Soak with 0.15mol / L pH = 6.5 phosphate buffer overnight, filtered
and reserved;
4.Wash and dry 2-3 fresh eggs, knocking broken egg head, and then
play a small hole in the bulk intake to collect egg white;
5.Filter egg white with sterile gauze, weighing the weight, and pre-
cooling in 4 ℃ refrigerator for more than 1h;
6. Page 6
6.Add the egg white to the treated resin (whichever is equivalent to
1/4 by weight of egg white) under constant stirring;
7.Vigorously stir (ice bath) and adsorption 2.5h, then 4 ℃ standing;
8.The next day, centrifuge at 3000 rpm for 15 min; collecting resin
and egg white;
9.First wash the rein with deionized water until the lotion is turbid;
10.And then use the same volume of 0.15mol / L pH6.5 phosphate
buffer stirring and washing for 15min in ice bath; repeat once again
and filter resin moisture at last;
7. Page 7
11.Soak the rein in an equal volume of 10% (NH4) 2SO4, stirred and
eluted, stirred for 30min each time and drained to collect the eluate.
Repeat the elution again, and combine the eluent;
12.Add 32g finely ground solid (NH4) 2SO4 to each 83mL eluent and add
it slowly with stirring. After (NH4) 2SO4 was completely dissolved, place
overnight at 4 ℃,
13.The next day, centrifuged at 1000rpm for 15min, and then collect the
precipitate;
14.Wash the precipitate twice with 1mL distilled water, and put into the
dialysis bag;
15.Place in 4 ℃ refrigerator, dialysis with distilled water for more than
24h, during which change the water for a couple of times;
8. Page 8
16.Take out the dialysis solution, adjust the pH to 4.6 with
0.1mol / L HCl, centrifuged, and collect the supernatant;
17.Add a small amount of distilled water to the precipitate ;
18.Adjust the pH to 5.5 with 1 mol / L NaOH, centrifuge,
discard the precipitate and collect the purified solution;
19.Put the purified solution into the dialysis bag, dry and
concentrate with research fine polyethylene glycol;
To be continued...
Collected by Creative BioMart.
9. Page 9
Thanks for watching!
Contact Creative BioMart
Address: 45-1 Ramsey Road Shirley, NY 11967, USA
Email: contact@creative-biomart.com
Website: http://www.creativebiomart.net
