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Prosedur *western blot* melibatkan beberapa langkah utama, yang jika digabungkan, menghasilkan identifikasi dan pengukuran protein yang akurat. Pertama, sampel yang berisi protein (misalnya, ekstrak sel atau jaringan) disiapkan. Protein kemudian dipisahkan berdasarkan ukuran menggunakan **elektroforesis gel poliakrilamida** (SDS-PAGE). Setelah pemisahan, protein dipindahkan ke membran, biasanya membran nitroselulosa atau PVDF, dalam proses yang disebut **blotting**. Membran kemudian diinkubasi dengan antibodi primer, yang spesifik terhadap protein target. Antibodi primer akan mengikat protein target yang ada pada membran. Setelah itu, membran diinkubasi dengan antibodi sekunder, yang mengenali antibodi primer. Antibodi sekunder ini biasanya terkonjugasi dengan enzim, seperti **horseradish peroxidase (HRP)**. Terakhir, membran diekspos terhadap substrat yang akan bereaksi dengan enzim (HRP), menghasilkan sinyal yang dapat dideteksi. Sinyal ini biasanya berupa pita yang terlihat pada film atau melalui sistem pencitraan digital, yang menunjukkan keberadaan protein target. Intensitas pita ini sebanding dengan jumlah protein target dalam sampel. Proses ini memungkinkan para ilmuwan untuk tidak hanya mendeteksi keberadaan protein, tetapi juga memperkirakan jumlahnya.
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