OPTIMASI TEKNIK WESTERN BLOT UNTUK DETEKSI EKSPRESI PROTEIN TANAMAN PADI (Oryza sativa L.)

. Susianti, Edi Sukmana, Ronny Lesmana, Unang Supratman

Abstract


Optimization of Western Blot Technique for Protein Expression of Rice Plant (Oryza sativa L.) 

Western blot (WB) technique has been widely used for analyzing protein expression and for identifying specific proteins derived from animals, plants, and microorganisms. During the use of WB, especially in agricultural studies, some difficulties are encountered such as unclear or unspecific protein bands, the presence of bubbles, and the absence of protein bands on membrane. This study aims to determine the WB conditions appropriate for the protein expression of rice plants (Oryza sativa L.). Protein from rice plant was extracted and the obtained protein lysate was then used for proteomic analysis using western blot with β-actin antibody. Our experiment showed that some optimized parameters like blocking buffers, the concentration of primary antibody and the ratio of secondary antibody determined the clarity of the results. β-actin was used as internal control that measured the success of the WB technique. Results showed that lysis process was important in determining good WB results in addition to the optimal blocking solution using a BSA of 0.2%, a primary antibody concentration of 1 μg mL1, and a secondary antibody of 1:10,000. Optimizing techniques during extraction, incubation, and documentation facilitated good WB results.

Keywords: β-actin; optimization; protein; rice plant; western blot


ABSTRAK

Teknik western blot (WB) telah banyak digunakan untuk analisis ekspresi protein dan mengidentifikasi protein spesifik dari hewan, tumbuhan dan mikroorganisme. Dalam implementasi teknik WB, khususnya studi dalam bidang pertanian, beberapa kesulitan ditemui seperti pita protein tidak jelas, tidak spesifik, adanya gelembung, hingga tidak munculnya pita protein pada membran. Penelitian ini bertujuan untuk mengetahui kondisi WB yang tepat untuk deteksi protein tanaman padi (Oryza sativa L.). Protein tanaman padi diekstraksi, kemudian lysate protein yang didapat dianalisis dengan metode westernblot menggunakan antibody β-actin. Penelitian kami menunjukkan bahwa beberapa parameter yang dioptimasi seperti larutan blocking, konsentrasi antibodi primer dan rasio antibodi sekunder akan menentukan hasil yang jelas. β-actin digunakan sebagai kontrol internal yang menjadi tolok ukur keberhasilan teknik WB. Hasil menunjukkan bahwa proses lisis menjadi hal penting dalam menentukan hasil WB yang baik disamping larutan blocking yang optimal menggunakan BSA 0,2%, konsentrasi antibodi primer 1 µg mL–1 dan antibodi sekunder 1:10.000. Mengoptimalkan teknik selama ekstraksi, inkubasi dan dokumentasi membantu mendapatkan hasil WB yang baik.


Keywords


β-actin; optimasi; protein; tanaman padi; western blot

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References


Alegria-Schaffer A, Lodge A, Vattern K (2009) Performing and optimizing western blots with an emphasis on chemiluminescent detection. Methods Enzymol 463:573–599. doi: 10.1016S0076-6879(09)63033-0

Ariyarathna HA, Oldach KH, Francki MG (2016) A comparative gene analysis with rice identified orthologous group II HKT genes and their association with Na+ concentration in bread wheat. BMC Plant Biol 16:21. doi: 10.1186/s12870-016-0714-7

Bass JJ, Wilkinson DJ, Rankin D, Phillips BE, Szewczyk NJ, Smith K, Atherton PJ (2017) An overview of technical considerations for western blotting applications to physiological research. Scand J Med Sci Sports 27:4–25. doi: 10.1111/sms.12702

Chatterjee M, Gupta S, Bhar A, Das S (2012) Optimization of an efficient protein extraction protocol compatible with two-dimensional electrophoresis and mass spectrometry from recalcitrant phenolic rich roots of chickpea (Cicer arietinum L.) Int J Proteomics 2012:1–10. doi: 10.1155/2012/536963

Cima-Cabal MD, Vazquez F, de Los Toyos JR, Del Mar García-Suárez M (2019) Protein expression analysis by western blot and protein–protein interactions. Methods Mol Biol 1968:101–111. doi: 10.1007/978-1-4939-9199-0_9

Esa NM, Ling TB, Peng LS (2013) By-products of rice processing: An overview of health benefits and applications. J Rice Res 1:107. doi: 10.4172/ jrr.1000107

Esteves CV, de Campos WG, de Souza MM, Lourenço SV, Siqueira WL, Lemos-Júnior CA (2019) Diagnostic potential of saliva proteome analysis: A review and guide to clinical practice. Braz Oral Res 33:1–13. doi:10.1590/1807-3107bor-2019.vol33.0043 

Feckova B, Kimáková P, Ilkovičová L, Szentpéteriová E, Debeljak N, Solárová Z,  Sačková V, Šemeláková M, Bhide M, Solár P (2016) Far-western blotting as a solution to the non-specificity of the anti-erythropoietin receptor antibody. Oncol Lett 12:1575–1580. doi:10.3892/ol.2016.4782

Ghosh R, Gilda JE, Gomes AV (2014) The necessity of and strategies for improving confidence in the accuracy of western blots. Expert Rev Proteomics 11:549–560. doi: 10.1586/14789450.2014.939635

Gibbons J (2014) Western blot: Protein transfer overview. North Am J Med Sci 6:158–159. doi:10.4103/1947-2714.128481

Gilda JE, Gomes AV (2013) Stain-free total protein staining is a superior loading control to β-actin for western blots. Anal Biochem 440:186–188. doi: 10.1016/j.ab.2013.05.027

Goldman A, Harper S, Speicher DW (2016) Detection of proteins on blot membranes. Curr Protoc Protein Sci 86:1–11. doi: 10.1002/cpps.15

Gorr TA, Vogel J (2015) Western blotting revisited: Critical perusal of underappreciated technical issues. Proteomics Clin Appl 9:396–405. doi: 10.1002/prca.201400118

Gulcicek EE, Colangelo CM, McMurray W, Stone K, Williams K, Wu T, Zhao H, Spratt H, Kurosky A, Wu B (2005) Proteomics and the analysis of proteomic data: An overview of current protein-profiling technologies. Curr Protoc Bioinformatics 13:1–40. doi: 10.1002/ 0471250953. bi1301s10

Huang YT, van der Hoorn D, Ledahawsky LM, Motyl AAL, Jordan CY, Gillingwater TH, Groen EJN (2019) Robust comparison of protein levels across tissues and throughout development using standardized quantitative western blotting. J Vis Exp 146:e59438. doi:10.3791/59438

Kurien BT, Scofield RH (2015) Western blotting: Methods and protocols. Methods in Molecular Biology Vol 1312. Springer Science-Business Media, New York

Lesmana R, Hanna G (2017) Fisiologi molekuler seri prosedur dan protokol laboratorium western blot. Fakultas Kedokteran Universitas Padjadjaran, Jatinangor

Li Q, Liu H, Du D, Yu Y, Ma C, Jiao F, Yao H, Lu C, Zhang W (2015) Identification of novel laminin- and fibronectin-binding proteins by far-western blot: Capturing the adhesins of Streptococcus suis type 2. Front Cell Infect Microbiol 5:82. doi: 10.3389/fcimb.2015.00082 

Li R, Shen Y (2013) An old method facing a new challenge: Re-visiting housekeeping proteins as internal reference control for neuroscience research. Life Sci 92:747–751. doi: 10.1016/j.lfs.2013.02.014

Li X, Bai H, Wang X, Li L, Cao Y, Wei J, Liu Y, Liu L, Gong X, Wu L, Liu S, Liu G (2011) Identification and validation of rice reference proteins for western blotting. J Exp Bot 62:4763–4772. doi: 10.1093/jxb/err084

Lin D-G, Wang C-S (2014) Extraction of total proteins from rice plant. Bio-protocol 4:e1277. doi: 10.21769/BioProtoc.1277

Luo H, Rankin GO, Straley S, Chen YC (2011) Prolonged incubation and stacked film exposure improve sensitivity in western blotting. J Pharmacol Toxicol Methods 64:233–237. doi: 10.1016/j.vascn.2011.06.001

Mahmood T, Yang P-C (2012) Western blot: Technique, theory, and trouble shooting. North Am J Med Sci 4:429–434. doi: 10.4103/1947-2714.100998

Mishra M, Tiwari S, Gomes AV (2017) Protein purification and analysis: Next generation western blotting techniques. Expert Rev Proteomics 14:1037–1053. doi: 10.1080/14789450.2017.1388167

Nie X, Li C, Hu S, Xue F, Kang YJ, Zhang W (2017) An appropriate loading control for western blot analysis in animal models of myocardial ischemic infarction. Biochem Biophys Rep 12:108–113. doi: 10.1016/j.bbrep.2017.09.001

Niu L, Yuan H, Gong F, Wu X, Wang W (2018) Protein extraction methods shape much of the extracted proteomes. Front Plant Sci 9:802. doi: 10.3389/fpls.2018.00802

Petras M, Drgova A, Kovalska M, Tatarkova Z, Tothova B, Krizanova O, Lehotsky J (2017) Effect of hyperhomocysteinemia on redox balance and redox defence enzymes in ischemia–reperfusion injury and/or after ischemic preconditioning in rats. Cell Mol Neurobiol 37:1417–1431. doi: 10.1007/s10571-017-0473-5

Rodrigues EP, Torres AR, da Silva Batista JS, Huergo L, Hungria M (2012) A simple, economical and reproducible protein extraction protocol for proteomics studies of soybean roots. Genet Mol Biol 35:348–352. doi: 10.1590/S1415-47572012000200016

Schurer JM, Nishimwe A, Hakizimana D, Li H, Huang Y, Musabyimana JP, Tuyishime E, MacDonald LE (2019) A one health systematic review of diagnostic tools for Echinococcus multilocularis surveillance: Towards equity in global detection. Food Waterborne Parasitol 15:1–24. doi:10.1016/j.fawpar.2019.e00048

Taylor SC, Posch A (2014) The design of a quantitative western blot experiment. BioMed Res Int 2014:361590. doi: 10.1155/2014/361590

Vavilis T, Delivanoglou N, Aggelidou E, Stamoula E, Mellidis K, Kaidoglou A, Cheva A, Pourzitaki C, Chatzimeletiou K, Lazou A, Albani M, Kritis A (2015) Oxygen–glucose deprivation (OGD) modulates the unfolded protein response (UPR) and inflicts autophagy in a PC12 hypoxia cell line model. Cell Mol Neurobiol 36:701–712. doi: 10.1007/s10571-015-0250-2

Vilhena MB, Franco MR, Schmidt D, Carvalho G, Azevedo RA (2015) Evaluation of protein extraction methods for enhanced proteomic analysis of tomato leaves and roots. An Acad Bras Cienc 87:1853–1863. doi: 10.1590/0001-3765201520150116

Villafanez F, Gottifredi V, Soria G (2019) Development and optimization of a miniaturized western blot-based screening platform to identify regulators of post-translational modifications. High Throughput 8:E15. doi:10.3390/ht8020015 

Walentowicz-Sadlecka M, Dziobek K, Grabiec M, Sadlecki P, Walentowicz P, Mak P, Szymankiewicz M, Kwinta P, Dutsch-Wicherek M (2018) The analysis of human leukocyte antigen-G level in patients with endometrial cancer by western blot technique. Am J Reprod Immunol 81:1–10. doi:10.1111/aji.13070

Wang W, Tai F, Chen S (2008) Optimizing protein extraction from plant tissues for enhanced proteomics analysis. J Sep Sci 31:2032–2039. doi: 10.1002/jssc.200800087

Wu J, Lee DY, Wang Y, Kim ST, Baek S-B, Kim SG, Kang KY (2014) Protein profiles secreted from phylloplane of rice leaves free from cytosolic proteins: Application to study rice-Magnaporthe Oryzae interactions. Physiol Mol Plant Pathol 88:28–35. doi: 10.1016/j.pmpp.2014. 08.003

Zhang C, Li H, Wang J, Zhang B, Wang W, Lin H, Luan S, Gao J, Lan W (2017a) The rice high-affinity K+ transporter OsHKT2;4 mediates Mg2+ homeostasis under high-Mg2+ conditions in transgenic Arabidopsis. Front Plant Sci 8:1823. doi: 10.3389/fpls.2017.01823

Zhang Y, Tang L, Liu X, Liu L, Cao W, Zhu Y (2017b) Modeling the leaf angle dynamics in rice plant. PLoS One 12:e0171890. doi: 10.1371/journal.pone.0171890

Zhao L, Liu C, Sun Y, Ban L (2012) A rapid and simplified method for protein silver staining in polyacrylamide gels. Electrophoresis 33:2143–2144. doi: 10.1002/elps.201200107




DOI: https://doi.org/10.29122/jbbi.v6i2.3249

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