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