BAB 5 KESIMPULAN DAN SARAN
5.2 Saran
Perlu diadakan analisis lebih lanjut pada pita-pita hasil pemisahan SDS PAGE dengan menggunakan LCMS sehingga dapat diketahui urutan rantai asam amino pada masing-masing pita tersebut.
38
DAFTAR PUSTAKA
Advansta corporation, protein analysis electrophoresis, bolting, and immunodetection., didownload dari http://advansta.com/PA_Guide.pdf
tanggal 6 Juli.
al-Janabi, J., J. A. Hartsuck, et al. 1972. "Kinetics and mechanism of pepsinogen activation." J Biol Chem 247: 4628-32.
Anonim, 2001, SDS Page Gel Electrophoresis, didownload dari https://ww2.chemistry.gatech.edu/~lw26/bCourse_Information/4581/techni ques/gel_elect/page_protein.html tanggal 27 maret.
Anonim, Polyacrylamide gel electrophoresis, didownload dari
http://upload.wikimedia.org/wikipedia/commons/4/46/SDS-PAGE_Electrophoresis.png tanggal 27 maret
Ansel, Howard C. 2005. Pengantar Bentuk Sediaan Farmasi Ed 4th.UI PRESS: Jakarta.
Azira, T., Amin. I., and Che Man, Y. B., 2012. Differentiation of bovine and porcine gelatins in processed products via Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and principal component analysis (PCA) techniques. International Food Research Journal 19 (3): 1175-1180.
Christensen, K. A., V. B. Pedersen, et al. 1977. "Identification of an enzymatically active intermediate in the activation of porcine pepsinogen." FEBS Lett 76: 214-8.
Davies, D. R. 1990. "The structure and function of the aspartic proteinases." Annu Rev Biophys Biophys Chem 19: 189-215.
Doi, H., Watanabe, E., Shibata, H., Tanabe, S. 2009. A reliable enzyme linked immunosorbent assay for the determination of bovine and porcine gelatin in processed foods. Journal of Agricultural and Food Chemistry. 57: 1721-6. Freifelder, David. 1987. Molecular Biology, 2nd edition. Boston: Jones and
Barlett
Fujinaga, M., M. M. Chernaia, et al. 1995. "Crystal structure of human pepsin and its complex with pepstatin." Protein Sci 4: 960-72.
Gelatin manufacturers institute of America, 2012, gelatin handbook, didownload dari http://www.gelatin-gmia.com/images/GMIAGelatin_Manual_2012.pdf
tanggal 23 Maret.
Gomez-Guillen, M. C., Perez-Mateos, M., Gomez-Estaca, J., Lopez-Caballero, E., Gimenez, B., & Montero, P. 2009. Fish gelatin: a renewable material for developing active biodegradable films. Trends in Food Science & Technology, Vol. 20, No. 1, pp. (3-16)
Gorgieva, S., Kokol, V. 2011. Collagen- vs. Gelatine-Based Biomaterials and Their Biocompatibility: Review and Perspectives, Biomaterials Applications for Nanomedicine, Prof. Rosario Pignatello (Ed.), ISBN: 978-953-307-661-4
Guo, T., Zhao, J., Chang, J., Ding, Z., Hong, H., Chen, J. & Zhang, J. 2006. Porous chitosan gelatin scaffold containing plasmid DNA encoding transforming growth factor-α1 for chrondrocytes proliferation.
39
Hafidz, R. N., Yaakob, C. M., Amin, I. and Noorfaizan, A., 2011, Chemical and functional properties of bovine and porcine skin gelatin, International Food Research Journal 18: 813-817.
Hemes, B.D.1998.Gel Electrophoresis of proteins. Oxford university press. New York.
Hermanto. S., sumarlin. L. O., Fatimah.W., 2013. Differentiation of Bovine and Porcine Gelatin Based on Spectroscopic and Electrophoretic Analysis
Journal food pharmaceutical sciences. 68-73.
Hidaka, S. & S. Y. Liu. 2002. Effect of gelatins on calcium phosphate precipitation: a possible application for distinguishing bovine bone gelatin from porcine skin gelatin. Journal of Food Composition and Analysis 16: 477-483.
James, M. N. and A. R. Sielecki 1986. "Molecular structure of an aspartic proteinase zymogen, porcine pepsinogen, at 1.8 A resolution." Nature
319(6048): 33-8.
James, M. N., A. Sielecki, et al. 1982. "Conformational flexibility in the active sites of aspartyl proteinases revealed by a pepstatin fragment binding to penicillopepsin." Proc Natl Acad Sci U S A 79: 6137-41.
Kageyama, T. and K. Takahashi (1983). "Occurrence of two different pathways in the activation of porcine pepsinogen to pepsin." J Biochem 93: 743-54. Kageyama, T. and K. Takahashi. 1987. "Activation mechanism of monkey and
porcine pepsinogens A. One-step and stepwise activation pathways and their relation to intramolecular and intermolecular reactions." Eur J Biochem 165: 483-90.
Murray, Robert K.dkk. 2006. Biokimia Harper Edisi 27.Penerbit Buku Kedokteran: Jakarta.
Nemati, M; Oveisi, M. R.; Abdollahi, H. and Sabzevari, O. 2004. Differentiation of bovine and porcine gelatins using principal component analysis. Journal of Pharmaceutical and Biomedical Analysis 34: 485-492
Rabadiya B, Rabadiya P. 2013. “a review: capsule shell material form gelatin to non animal origin material.” IJPRBS 2(3):42-71.
Raraswati, M.A., Triyana. K., Triwahyudi, and Rohman. A., 2013, Defferentiation of Bofine and Porcine in soft candy based on amino acid profiles and chemometrics. Journal food pharmaceutical sciences 1-6.
Schriber, L.A, & C. J. Moore. 2002. Gelatine Handbook. Wiley VCH Verlag GmbH & Co. Biocentennial.
Sielecki, A. R., A. A. Fedorov, et al. 1990. "Molecular and crystal structures of monoclinic porcine pepsin refined at 1.8 A resolution." J Mol Biol 214: 143-70.
Syamsuni, Haji. 2005. Farmasetika dasar dan hitungan farmasi. Penebit Buku Kedokteran EGC : Jakarta.
The Gelatin Manufacturers Institute of America, (GMIA) 2011, Raw Materials, Production & Uses of Gelatin, didownload dari http://www.gelatin-gmia.com/html/rawmaterials_app.html, 27 Agiustus 2011.
Venien, A., & Levieux, D. 2005. Differentiation of bovine from porcine gelatins using polyclonal anti-peptide antibodies in indirect and competitive indirect ELISA. Journal of Pharmaceutical and Biomedical Analysis, 39, 418–424.
40
Westermeier, 2004. Electrophoresis in Practice: A Guide to Theory and Practice. New-Jersey: John Wiley & Sons inc.
Wilson K, Walker J. 2000. Principles and Techniques of Practical Biochemistry Fifth Edition. United Kingdom: Cambridge University Press.
Wu, Y., S. Kaveti, et al. (2006). "Extensive deuterium back-exchange in certain immobilized pepsin columns used for H/D exchange mass spectrometry."
Anal Chem 78: 1719-23.
Zhang, G.F., Liu, T., Wang, Q., Chen, L., Lei, J., Luo, J., Ma, G. and Su, Z. 2009. Mass spectrometric detection of marker peptides in tryptic digests of gelatin: a new method to differentiate between bovine and porcine gelatin.
Food Hydrocolloids. 23: 2001–2007.
Zhang, G.F., Liu, T., Wang, Q., Lei, J.D., Ma, G.H. and Su, Z.G. 2008. Identification of marker peptides in digested gelatins by high performance liquid chromatography/mass spectrometry. Chinese Journal of Analytical Chemistry. 36: 1499–504.
Zhang, Z. and D. L. Smith .1993. "Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation." Protein Sci 2: 522-31.
LAMPIRAN
Lampiran 1 Alur Penelitian
42
Lampiran 2 Foto Penelitian
Kaset dan rak elektroforesis Pemanasan sampel sebelum dielektroforesis
Chamber dan adaptor elektroforesis Proses stainning gel
Vortex ekstrak gelatin Ekstrak gelatin
Hasil ekstrak gelatin Tes kualitatif keberadaan protein hasil ekstraksi
43
Lampiran 3 Preparasi Reagent SDS-PAGE
a. Larutan Stok Acrylamide/Bis (30% T; 2,67% C)
29,2 g akrilamid dilarutkan dalam 100 ml air deionisasi, kemudian ditambahkan 0,8 ml N’N’-bis-methylene-acrylamide ke dalam larutan aduk hingga larut dengan stirer, kemudian larutan disaring dan disimpan pada suhu 4oC di tempat yang terhindar dari cahaya, larutan dapat disimpan maksimal 30 hari sebelum digunakan.
b. SDS 5% (w/v)
5 g SDS dilarutkan dalam 90 ml air deionisasi diaduk dengan hati-hati kemudian ditambakan air deionisasi hingga 100 ml.
c. 1,5 M Tris-HCl; pH 8,8
18,15 g Basa Tris dilarutkan dalam 80 ml air deionisasi diaduk dengan hati-hati kemudian pH disesuaikan hingga 8.8 dengan penambahan 6 N HCl. kemudian air deionisasi ditambahkan pada larutan hingga 100 ml, larutan di simpan pada suhu 4oC.
d. Sampel Buffer (0,5 M Tris-HCl; pH 6,8)
6 g Basa Tris dilarutkan dalam 60 ml air deionisasi diaduk dengan hati-hati kemudian pH disesuaikan hingga 6,8 dengan penambahan 6 N HCl. Kemudian ditambahkan air deionisasi hingga 100 ml. larutan disimpan pada 4oC.
e. Runing Buffer (SDS reducing buffer)
1,25 ml stacking buffer; 2,5 ml glyserol, 2 ml 10% SDS; dan 0,2 ml 0,5% (w/v) bromphenol blue ditambahkan dalam 3,55 ml air deionisasi. Air deionisasi ditambahkan hingga volume total 9,5 ml, larutan disimpan pada suhu ruang. Larutan digunakan dengan menambahkan 50 ml β–mecaptoethanol ke dalam 950 ml sample
buffer sebelum digunakan, encerkan sample paling sedikit 1:2 di dalam sample buffer dan panaskan 95oC selama 4 menit.
f. 10x Buffer Elektroda
30,3 gr Basa Tris; 144 g glisin; dan 10 g SDS dilarutkan dalam 100 ml air deionisasi, larutan diaduk kemudian ditambahkan air deionisasi
44
hingga volume total 1000 ml. Larutan disimpan pada 4oC dan dihangatkan hingga suhu ruangan sebelum digunakan.
g. 10% APS(disiapka segar sebelum pemakaian)