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APPENDICES
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APPENDIX A
List of chemical and materials
Chemicals & Materials Company
Acetonitrile (CH3CN) ACROS Organics, USA
Agarose Amersham Biosciences, USA
40% Acrylamide: Bisacrylamide (37.5:1) Bio-Rad, USA Amicon centrifugal tube MWCO 3 kDa Milipore, USA Ammonium bicarbonate (NH4)HCO3 Sigma, USA
Ammonium persulfate ((NH4)2S2O8) Pharmacia Biotech, Sweden Anti-human GM2 activator protein
(rabbit IgG; ab107501) Abcam, UK Anti-human GM2 activator protein
(rabbit IgG; ab118433) Abcam, UK
biotinylated labeled Aleuria aurantia lectin (AAL) Vector Laboratories, USA
Bovine serum albumin Pierces, USA
Bromophenol blue (C19H10Br4O5S) Pharmacia Biotech, Sweden
C18 ZipTip® Milipore, USA
Carrier ampholytes (pH 3-5.6) Amersham Biosciences, USA Carrier ampholytes (pH 4-7) Amersham Biosciences, USA 3,3-diaminobenzidine (DAB) Sigma, USA
3-[(3-cholamidopropyl)-dimethylammonio]-1-
propanesulfonic acid (CHAPS; C32H58N2O7S) J.T.Baker, USA Chymotrypsin (sequencing grade) Promega, USA Dithiothreitol, DTT (C4H10O2S2) Sigma, USA Dimethyl Sulfoxide, DMSO [(CH3)2SO)] Sigma, USA
DryStrip cover fluid Amersham Biosciences, USA
ECLTM kit Amersham Biosciences, USA
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Chemicals & Materials Company
Enzyme-linked Immunosorbent Assay Kit
for GM2 Ganglioside Activator (GM2A) Uscn Life Science, UK Ethanol, absolute (C2H5OH) Sigma, USA
Filter paper Millipore, USA
Formic acid (CH2O2) Sigma, USA
Glycine (NH2CH2COOH) Sigma, USA
Glycerol [HOCH2CH(OH)CH2OH] Riedel-de Haën, USA
Hematoxylin Sigma, USA
HRP-conjugated secondary antibody Abcam, UK
HRP-conjugated streptavidin Vector Laboratories, USA Hydrochloric acid (HCl) J.T.Baker, USA
Hydrogen peroxide (H2O2) Merck, Germany
Immobiline™ Dry strips length 18 cm; pH 3-5.6 Amersham Biosciences, USA Immobiline™ Dry strips length 18 cm; pH 4-7 Amersham Biosciences, USA Immobilon-P transfer membrane
(PVDF transfer membrane) Millipore, USA Iodoacetamide , IAA (C2H4INO) Fluka, Switzerland
Low molecular weight protein markers Pharmacia Biotech, Sweden
Methanol (MeOH) Sigma, USA
β-Mercaptoethanol (HSCH2CH2OH) Merck, Germany
Midi GeBAflex-tube MWCO 3 kDa Gene Bio-Application, Israel PD-10 desalting column Amersham Biosciences, USA Phosphate buffer saline (PBS) Sigma, USA
Polyxyethylene sorbitan monolaurate (Tween 20) Sigma, USA
Protein assay kit Bio-Rad, USA
Sodium azide (NaN3) Sigma, USA
Sodium bicarbonate (NaHCO3) Sigma, USA
Sodium chloride (NaCl) Merck, Germany
Sodium di-hydrogen phosphate (NaH2PO4) Sigma, USA Sodium dodecyl sulfate, SDS (C12H25SO4Na) Bio-Rad, USA
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Chemicals & Materials Company
di-Sodium hydrogen phosphate (Na2HPO4) Sigma, USA Sodium hydroxide (NaOH) Merck, Germany SYPRO® Ruby gel staining Molecular Molecular Probes, USA N, N, N', N'-Tetramethylethylenediamine (TEMED) Merck, Germany Thiourea (NH2CSNH2) Riedel-de Haën, USA Trifluoroacetic acid (CF3COOH) Sigma, USA
Tris-[hydroxymethyl]-aminomethane
(NH2C(CH2OH)3) Merck, Germany Trypsin (sequencing grade) Promega, USA
Urea (NH2CONH2) J.T.Baker, USA
76
APPENDIX B
List of instruments
Instruments Company
Analytical balance 2 digits Mettler Toledo, USA Analytical balance 6 digits Mettler Toled, USA
Centrifuge Beckman Coulter, USA
ELISA reader Model MRX II Dynex Technologies, USA Fujifilm LAS-4000 Luminiescent Image Analyzer Fujifilm, Japan
Gel caster (mini gel, 8 × 10 cm) Bio-Rad, USA Gel caster (2D gel, 20 × 20 cm) Bio-Rad, USA
Hot plate stirrer Whatman, USA
IEF apparatus (IPGphore III) Amersham Biosciences, USA ImageMaster TM 2D Platinum software version 5.0 Amersham Biosciences, Sweden ImageScope softwere Aperio Technologies, USA
IPG strip holder Amersham Biosciences, USA
Lyophilizer Kingmech, Taiwan
NanoLC-MS/MS Thermo Fisher Scientific, USA
MALDI-Q-TOF-MS Bruker Daltonik GmbH, Germany
MALDI-TOF-MS Bruker Daltonik GmbH, Germany
MALDI-TOF-TOF-MS Applied Biosystems, USA
pH meter Metronm, Switzerland
Protein blotting apparatus Amersham Biosciences, USA Power supply (PowerPac 1000) Bio-Rad, USA
Electrophoresis running tank Bio-Rad, USA
Sonicator Model 2510 Branson, USA
SpeedVac concentrator Labogene, Denmark
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Instruments Company
SPSS softwere version 17.0 Chicago, USA
Typhoon 9200 image scanner Amersham Biosciences, USA
Vortex Genie 2 Scientific Industries, USA
78
APPENDIX C
Preparation of reagents and buffer
Separating gel composition
Reagents Volume required for
10% 12.50% 15%
40% Acrylamide:Bisacrylamined
(37.5:1) 5.00 mL 6.25 mL 7.50 mL
1.5 M Tris-HCl (pH 8.8) 5.00 mL 5.00 mL 5.00 mL
Milli-Q water 9.70 mL 8.45 mL 7.20 mL
10% (v/v) SDS 200 µL 200 µL 200 µL
10% (w/v) ammonium persulphate 100 µL 100 µL 100 µL
TEMED 10 µL 10 µL 10 µL
Total volume 20 mL 20 mL 20 mL
Stacking gel composition (4% gel)
Reagents Volume required for
40% Acrylamide:Bisacrylamide
(37.5:1) 0.50 mL
1.5 M Tris-HCl (pH 8.8) 1.25 mL
Milli-Q water 3.15 mL
10% (v/v) SDS 50 μL
10% (w/v) ammonium persulphate 50 μL
TEMED 10 μL
Total volume 5 mL
79 Denaturing loading buffer (for SDS-PAGE)
Reagents Final concentrations
Tris-HCL pH 6.8 50 mM
SDS 2% (w/v)
Glycerol 10% (v/v)
ß-mercaptoethanol 5% (v/v)
Bromophenol blue trace
Store 1 mL aliquots at -20°C for 6 months
Lysis buffer
Reagents Final concentrations
Urea 7 M
Thiourea 2 M
CHAPS 4% (w/v)
Bromophenol blue trace
DTE 65 mM
Ampholyte (IPG buffer) 0.5-2 %(v/v)
Store 1 mL aliquots at -20°C for 6 month, DTE and ampholyte added before using
Do not re-freeze after defrosting
Equilibrium buffer
Reagents Final concentrations
1.5 M Tris-HCl, pH 8.8 50 mM
Urea 6 M
Glycerol 30% (w/v)
SDS 2% (w/v)
Bromophenol blue trace
Store at room temperature
80
Separating gel composition (For preparing the gradient gel 20 cm × 20 cm × 1.5 mm, ~60 mL per gel)
Reagents Volume required for
10% 12.50% 15%
40% Acrylamide:Bisacrylamide
(37.5:1) 75 mL 112.5 mL 135 mL
1.5 M Tris-HCl (pH 8.8) 75 mL 75 mL 75 mL
Milli-Q water 150 mL 112.5 mL 90 mL
10% (w/v) ammonium persulphate 750 μL 750 μL 750 μL
TEMED 75 μL 75 μL 75 μL
Total volume 300 mL 300 mL 300 mL
Phosphate buffer saline with Tween (PBST)
Reagents Amount required for
Na2HPO4 1.44 g
KH2PO4 0.25 g
NaCl 8 g
KCl 0.25 g
MgCl2 1 g
Tween 20 0.05% (v/v)
Total volume is 1 l, store at room temperature
Running buffer
Reagents Final concentrations
Tris-base 25 mM
Glycine 192 mM
SDS 0.1 (w/w)
Store at room temperature
81 Transfer buffer
Reagents Final concentration
Tris-base 25 mM
Glycine 192 mM
Methanol 20% (v/v)
Store at room temperature
82
APPENDIX D
Clinical information of lung cancer patients
Table 2.1 Information of urine samples from Maharaj Nakron Chiang Mai Hospital, Chiang Mai, Thailand.
Sample no. Histology Gender Age Stage
L001 Adenocarcinoma M 51 IV
L002 Adenocarcinoma M 53 IV
L003 Small cell lung cancer M 60 IV
L004 Small cell lung cancer M 71 IV
L005 Small cell lung cancer M 72 IV
L006 Adenocarcinoma M 62 IV
L007 Adenocarcinoma M 43 IV
L008 Adenocarcinoma M 55 IV
L009 Adenocarcinoma M 51 IV
L010 Adenocarcinoma M 42 IV
L011 Adenocarcinoma M 59 IV
L013 Adenocarcinoma F 47 IV
L014 Small cell lung cancer F 56 IV
L015 Small cell lung cancer F 47 III
L016 Small cell lung cancer F 66 IV
L017 Other type of carcinoma F 54 IV
L018 Small cell lung cancer F 56 III
L019 Other type of carcinoma F 49 IV
L020 Adenocarcinoma M 56 IV
L022 Small cell lung cancer M 47 IV
L023 Adenocarcinoma F 62 IV
L024 Adenocarcinoma F 56 IV
L025 Adenocarcinoma F 54 IV
L026 Small cell lung cancer M 72 III
L027 Adenocarcinoma M 61 IV
L028 Small cell lung cancer F 53 IV
83 Table 2.1 (Continue).
Sample no. Histology Gender Age Stage
L029 L030
Adenocarcinoma Adenocarcinoma
F M
31 53
IV IV
L031 Other type of carcinoma M 28 IV
L032 Small cell lung cancer F 50 IV
L033 Small cell lung cancer M 74 IV
L034 Small cell lung cancer M 56 IV
L035 Other type of carcinoma M 42 IV
L036 Small cell lung cancer M 69 IV
L037 Adenocarcinoma M 69 III
L038 Adenocarcinoma M 63 III
L039 Adenocarcinoma M 53 IV
L040 Adenocarcinoma M 57 IV
L041 Squamous cell carcinoma M 57 IV
L042 Small cell lung cancer M 59 IV
L050 Squamous cell carcinoma M 56 IV
L051 Squamous cell carcinoma M 35 IV
L052 Squamous cell carcinoma M 31 IV
L053 Small cell lung cancer F 37 IV
L054 Adenocarcinoma M 42 IV
L055 Adenocarcinoma M 55 III
L056 Adenocarcinoma M 33 IV
L057 Small cell lung cancer F 45 IV
84
Table 2.2 Information of urine and serum samples from National Taiwan University Hospital, Taipei, Taiwan.
Sample no. Histology Gender Age Stage
5760275 Adenocarcinoma F 60 IA
1484156 Adenocarcinoma F 66 IA
L001 Squamous cell carcinoma F 52 IV
L002 Adenocarcinoma F 49 IV
L003 Adenocarcinoma M 49 IIIA
L004 Adenocarcinoma M 79 IV
L005 Adenocarcinoma M 49 IV
L006 Adenocarcinoma F 43 IV
L007 Adenocarcinoma M 42 IIIB
L008 Squamous cell carcinoma M 74 IIIB
L009 Adenocarcinoma F 80 IB
L010 Adenocarcinoma F 37 IV
L011 Squamous cell carcinoma F 55 IV
L012 Pleomorphic carcinoma M 30 IIIA
L013 Small cell lung cancer M 63 IV
L014 Adenocarcinoma M 42 IV
L015 Adenocarcinoma F 32 IA
L016 Adenocarcinoma M 81 IA
L017 Adenocarcinoma F 37 IV
L018 Adenocarcinoma M 72 IIB
L019 Small cell lung cancer M 68 IV
L020 Adenocarcinoma M 50 IV
L021 Adenocarcinoma M 53 IA
L022 Adenocarcinoma M 55 IA
L023 Adenocarcinoma M 53 IV
L024 Adenocarcinoma F 66 IIIB
L025 Non small cell lung cancer M 61 IIA
L026 Adenocarcinoma F 63 IV
L027 Adenocarcinoma F 75 IV
L028 Adenocarcinoma M 72 IV
L029 Adenocarcinoma F 55 IV
L030 Adenocarcinoma M 59 IIIA
L031 Non small cell lung cancer M 62 IIIB
L032 Adenocarcinoma F 49 IV
85 Table 2.1 (Continue).
Sample no. Histology Gender Age Stage
L033 Adenocarcinoma M 70 IV
L034 Adenocarcinoma F 60 IV
L035 Adenocarcinoma F 58 IV
L036 Adenocarcinoma F 59 IIIB
L037 Adenocarcinoma F 70 IIIA
L038 Adenocarcinoma F 31 IV
L039 Adenocarcinoma F 67 IV
L040 Adenocarcinoma M 66 IV
L041 Adenocarcinoma F 61 IIIA
L042 Adenocarcinoma M 77 IIIB
L043 Adenocarcinoma M 55 IV
L044 Adenocarcinoma F 44 IV
L045 Squamous cell carcinoma M 61 IV
L046 Small cell lung cancer M 84 IV
L047 Adenocarcinoma F 79 IV
L048 Adenocarcinoma F 79 IV
L049 Adenocarcinoma F 75 IIIB
L050 Squamous cell carcinoma M 72 IIIB
L051 Squamous cell carcinoma M 64 IV
L052 Adenocarcinoma F 61 IV
L053 Adenocarcinoma F 69 IA
L054 Adenocarcinoma F 51 IV
L055 Adenocarcinoma F 64 IA
L056 Adenocarcinoma M 58 IV
L057 Adenocarcinoma F 57 IV
L058 Adenocarcinoma M 55 IV
L059 Adenocarcinoma M 72 IV
L060 Adenocarcinoma F 73 IV
L061 Adenocarcinoma F 65 IB
L062 Adenocarcinoma F 61 IV
L063 Adenocarcinoma F 81 IA
L064 Adenocarcinoma M 42 IV
L065 Adenocarcinoma F 70 IV
L066 Non small cell lung cancer M 72 IIIB
L067 Adenocarcinoma M 61 IA
L068 Adenocarcinoma M 57 IV
86 Table 2.1 (Continue).
Sample no. Histology Gender Age Stage
L069 Adenocarcinoma F 76 IIA
L070 Adenocarcinoma F 71 IIIB
L071 Adenocarcinoma F 46 IV
L072 Small cell lung cancer M 80 IIIB
L073 Squamous cell carcinoma M 73 IIIA
L074 Adenocarcinoma M 68 IV
L075 Adenocarcinoma F 69 IA
L076 Adenocarcinoma M 78 IIIB
L077 Adenocarcinoma F 76 IV
L078 Adenocarcinoma M 52 IV
L079 Adenocarcinoma F 61 IB
L080 Adenocarcinoma M 46 IV
L081 Squamous cell carcinoma M 66 IV
L082 Squamous cell carcinoma M 76 IV
L083 Squamous cell carcinoma M 78 IIA
L084 Adenocarcinoma F 64 IB
L085 Adenocarcinoma F 66 IV
L086 Adenocarcinoma F 71 IV
L087 Carcinoma M 72 IV
L088 Adenocarcinoma F 56 IV
L089 Adenocarcinoma F 47 IIIA
L090 Adenocarcinoma F 63 IV
L091 Non small cell lung cancer F 53 IV
L092 Adenocarcinoma F 81 IV
L093 Adenocarcinoma M 61 IV
L094 Adenocarcinoma F 61 IV
L095 Adenocarcinoma M 63 IV
L096 Adenocarcinoma F 59 IV
L097 Adenocarcinoma M 33 IV
L098 Adenocarcinoma F 53 IV
L099 Adenocarcinoma F 61 IV
L100 Adenocarcinoma M 51 IV
L101 Adenocarcinoma M 63 IA
L102 Adenocarcinoma M 74 IV
L103 Adenocarcinoma M 57 IIIB
L104 Adenocarcinoma M 88 IIIA
Table 2.1 (Continue)
87
Sample no. Histology Gender Age Stage
L105 Adenocarcinoma F 72 IV
L106 Adenocarcinoma F 62 IB
L107 Adenocarcinoma M 56 IV
L108 Adenocarcinoma F 72 IIIA
L109 Adenocarcinoma F 60 IIIB
L110 Adenocarcinoma F 44 IV
L111 Non small cell lung cancer F 74 IIA
L112 Adenocarcinoma M 72 IIIB
L113 Adenocarcinoma M 70 IV
L114 Adenocarcinoma F 56 IIB
L115 Adenocarcinoma F 76 IV
L116 Squamous cell carcinoma F 54 IV
L117 Adenocarcinoma M 58 IV
L118 Adenocarcinoma M 70 IV
L119 Adenocarcinoma F 75 IV
L120 Adenocarcinoma F 70 IV
L121 Adenocarcinoma F 49 IB
L122 Non small cell lung cancer M 70 IV
L123 Adenocarcinoma F 46 IV
L124 Carcinoma F 50 IV
L125 Adenocarcinoma F 73 IV
L126 Adenocarcinoma M 70 IIIA
L127 Adenocarcinoma F 67 IV
L128 Adenocarcinoma F 77 IV
L129 Adenocarcinoma F 52 IV
L130 Adenocarcinoma F 49 IV
L131 Adenocarcinoma M 56 IV
88
APPENDIX E
PUBLICATIONS
1. Potprommanee L., Ma H.T., Chen C.H., Aobchey P., Boonyapranai K., Chewaskulyong B., Sangthong P., Shank L., Chen S.T. Human Urinary GM2- activator protein as a potential biomarker for lung cancer. J Proteomics Bioinform. 2013; 6: 264–270.
2. Potprommanee L., Boonyapranai K., Sangthong P., Chewaskulyong B., Chen ST., Shank L. Verification of GM2 activator protein for potential application as lung cancer biomarker. JOMB. 2014; 2301–3796: in press.
3. Potprommanee L., Ma H.T., Shank L., Juan Y.H., Liao W.Y., Chen S.T., Yu C.J. GM2 activator protein: a new biomarker for lung cancer. J Thorac Oncol.
2014; in press.
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CURRICULUM VITAE
Name Miss Laddawan Potprommanee
Date of birth 13 April 1986
Place of birth Chachoengsao Province Education
Mar 2009 B.Sc. (Biochemistry and Biochemical Technology), Faculty of Science, Chiang Mai University, Chiang Mai, Thailand May 2011 M.S. (Biotechnology) Graduate School, Chiang Mai
University, Chiang Mai, Thailand
July 2014 Ph.D. (Biotechnology), Graduate School, Chiang Mai University, Chiang Mai, Thailand
Employment history Nov 2011 – Jan 2014 Research assistant, Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
Oral Presentation
1. Potprommanee L., Tantipaiboonwong P., Aobchey P., Shank L., Chen S.T.
GM2-activator protein: A potential biomarker for lung cancer identified by urine proteome analysis. Urinomics 2013. September 9-11, 2013, Caparica Portugal.
2. Potprommanee L., Boonyapranai K., Sangthong P., Chewaskulyong B., Chen ST., Shank L. Verification of GM2 Activator protein for potential application as lung cancer biomarker. ICBBE 2014. August 26-27, 2014, Taipei, Taiwan.
144 Poster Presentation
1. Potprommanee L., Tantipaiboonwong P., Aobchey P., Shank L., Chen S.T.
Proteomic analysis of urinary protein markers in normal and lung cancer patients.
Taiwan Proteomics Society Annual conference: Disease Proteomics and Metabolomics. May 31-June 01, 2012, Kaoshiung Medical University, Taiwan.
Publications
1. Potprommanee L., Ma H.T., Chen C.H., Aobchey P., Boonyapranai K., Chewaskulyong B., Sangthong P., Shank L., Chen S.T. Human Urinary GM2- activator protein as a potential biomarker for lung cancer. J Proteomics Bioinform. 2013; 6: 264–270.
2. Potprommanee L., Boonyapranai K., Sangthong P., Chewaskulyong B., Chen ST., Shank L. Verification of GM2 activator protein for potential application as lung cancer biomarker. JOMB. 2014; 2301–3796: in press.
3. Potprommanee L., Ma H.T., Shank L., Juan Y.H., Liao W.Y., Chen S.T., Yu C.J. GM2 activator protein: a new biomarker for lung cancer. J Thorac Oncol.
2014; in press.
