Supplementary material Extraction, purification and characterization of mdpf




Дата канвертавання24.04.2016
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Title: Immunological adjuvant effect of the peptide fraction from the larvae of Musca domestica

Authors: Chen LQ, et al.


Supplementary material

Extraction, purification and characterization of MDPF

The third instar larvae of Musca domestica were collected in Zhejiang Xiangshan Nursery, China in November, 2010. A voucher specimen (No. 20101105) has been deposited at the Laboratory of Nature Drug, College of Animal Sciences, Zhejiang University, China. MDPF were isolated and purified from the larvae of Musca domestica as previously described (Guo ZY, Shi YH, Le GW. Adsorption and separa tion of Musca domestica antimicrobial peptide with macroporous adsorption resin. Natural Product Research and Development 2008;20: 969–973). Briefly, the collected fresh larva were washed with distilled water, dried with absorbent paper and then frozen to death at –20 °C. 300 g larva were weighed and homogenized with 600 ml of 3% acetic acid (HAc) for 5 min in a glass beaker using a high speed tissue gravity mill. The homogenate was stored overnight at 4 °C, centrifuged at 4000 rpm for 10 min at 4 °C. The resulting precipitate was treated once again with the same method. The combined supernatant was heated at 100 °C for 1 h, stored overnight at 4 °C for 3 h, and then filtered through filter paper. The filtrate was lyophilized to afford a total extract (26.27 g). The dried extract (17.50 g) was subjected to D101 resin column chromatography, washed with H2O, and eluted with 55% ethanol (EtOH). The collected eluates were concentrated and lyophilized to afford a peptide-enriched fraction (MDFP). Protein concentration in MDPF was determined using bicinchoninic acid (BCA) protein assay kit according to the manufacturer׳s instructions. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of MDFP was performed on 5.0% stacking gel and a 12.5% separation gel.


Title: Immunological adjuvant effect of the peptide fraction from the larvae of Musca domestica

Authors: Chen LQ, et al.


Supplementary Fig. 1.


1

2

3

M






250 kD

130 kD

100 kD

70 kD

55 kD

35 kD

25 kD

15 kD

10 kD

SDS-PAGE analysis of the peptide fraction from Musca domestica larvae (MDPF). Lane 1, MDPD (0.5 mg/ml); Lane 2, MDPD (1 mg/ml); Lane 3, MDPD (2 mg/ml); M, molecular mass marker.







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