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University оf Kragujevac, Faculty of Science
CENTRE FOR PRE-CLINICAL TESTING OF ACTIVE SUBSTANCES
LABORATORY FOR CELL AND MOLECULAR BIOLOGY
Radoja Domanovića 12, 34000 Kragujevac, Serbia
http://cpctas.pmf.kg.ac.rs
 e-mail: cpctas@kg.ac.rs
Number
User request
Ch-Pl/08
Milena Ćurčić, Faculty of
Science, University of
Kragujevac
Model system
Material reception /
Start of testing
September, 2011.
Responsible person
Dragana Đačić
Active substance
Analysis
Cisplatin,
Methanolic extract of
leaves from Ligustrum
Western Blot (UM.11)
vulgare
HCT-116
UM.01, UM.02, UM.03, UM.04
Methanolic extract of
fruit from Ligustrum
vulgare
Title
Determination of changes on protein profile and Western blotting detection of
protein in different treatment on HCT-116 cell line
The aim of this study was to determinate of changes on protein profile in HCT-116
Objective
cell line treated with cisplatin and methanolic extracts from leaves and fruit of
Ligustrum vulgare
Material,
Drugs
methods, patients HCT-116 cell line were treated with 2 ml 50 µM of cisplatin, 50 µg/ml methanolic
extract of leaves from Ligustrum vulgare, and 50 µg/ml methanolic extract of fruits
from Ligustrum vulgare for 24 h.
Cell preparation and culturing (UM.01, UM.02, UM03)
HCT-116 (human colon cancer) was obtained from American Type Culture
Collection. Cells were maintained in DMEM supplemented by 10% FBS, with 100
units/ml of penicillin and 100 µg/ml of streptomycin. Cells were cultured in a
humidified atmosphere of 5% CO2 at 37 °C. Cells were growth in 75 cm2 culture
bottles supplied with 15 ml DMEM, and after a few passages cells were seeded in
6-well plate (exponentially growing viable cells were used throughout the assay).
Western blot (UM.11)
HCT-116 cell line were seeded in 6-well plate (100 000 cells per well). After 48 h of
cells incubation, the medium was replaced with 2 ml of medium containing 50 μM
concentrations of cisplatin, 50 µg/ml methanol extract of leaves and 50 µg/ml
methanol extract of fruit from Ligustrum vulgare. Untreated cells served as a
control. After 24 h of treatment we were lyses cells and determinate of
concentration of proteins. We were measure concentration of proteins by
Lowry method. After determination of protein concentration we prepared gel
for separating proteins by electrophoresis. After electrophoresis we were
putted gel and nitrocellulose membrane in “sandwich”.
Sandwich: Black/cathode = 1sponge + 1Watmann + Gel + Nitrocellulos
membrane + 1 Watmann + 1 sponge = Red/anode. After 1 h protein
migration on membrane. We added primary antibody for marking protein.
Incubation period was 1h. After washing we were putted secondary HRPDragana Đačić, MSci
Snežana Marković, PhD
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labeled antibody. For detection of marking protein we mixed ECL kit: 1 mL
solution A + 1 mL solution B and spread 300 µL of mixture on membrane.
Detection was done at 428nm.
Results
Discussion
Conclusion
References
Notes
Blake, M.S., et al. (1984). A Rapid, Sensitive Method for Detection of Alkaline
Phosphatase Conjugated Antibody on Western Blots. Anal. Biochem., 136:175-178.
This report applies only to the tested substances. Responsible for the report,
(accuracy and technical explanations of results) are researcher and manager and are
considered the report's authors, which they have confirmed with their signature. The
report should not be used or reproduced partially, except in its entirety in form of
the publication of results as an integral part of the report. Publication of results
based on this report must be approved by the authors.
Sign
Responsible person for testing
Dragana Đačić
Date
20.10.2011.
Responsible person for Laboratory
Dr Snežana Marković
Figure 1. Nitrocellulose membrane
Dragana Đačić, MSci
Snežana Marković, PhD