The purpose of the protein assay is to determine the amount or concentration of a specific protein or array of proteins in a sample. There are two main steps: protein extraction and quantification. For extraction, methods are used to efficiently extract proteins from biological material and separate them from other components. For quantification, spectroscopic, colorimetric, and fluorescent dye-based methods can be used to detect and measure the amount of protein present. The choice of assay depends on compatibility with the sample type and components, required sample volume, speed, and available equipment.
The purpose of the protein assay is to determine the amount or concentration of a specific protein or array of proteins in a sample. There are two main steps: protein extraction and quantification. For extraction, methods are used to efficiently extract proteins from biological material and separate them from other components. For quantification, spectroscopic, colorimetric, and fluorescent dye-based methods can be used to detect and measure the amount of protein present. The choice of assay depends on compatibility with the sample type and components, required sample volume, speed, and available equipment.
The purpose of the protein assay is to determine the amount or concentration of a specific protein or array of proteins in a sample. There are two main steps: protein extraction and quantification. For extraction, methods are used to efficiently extract proteins from biological material and separate them from other components. For quantification, spectroscopic, colorimetric, and fluorescent dye-based methods can be used to detect and measure the amount of protein present. The choice of assay depends on compatibility with the sample type and components, required sample volume, speed, and available equipment.
The purpose of the protein assay is to determine the amount or concentration of a specific protein or array of proteins in a sample. There are two main steps: protein extraction and quantification. For extraction, methods are used to efficiently extract proteins from biological material and separate them from other components. For quantification, spectroscopic, colorimetric, and fluorescent dye-based methods can be used to detect and measure the amount of protein present. The choice of assay depends on compatibility with the sample type and components, required sample volume, speed, and available equipment.
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Protein assay
CHRISTA EDO G0017044 recall tujuan
The purpose of the protein assay is to determine the amount or
concentration of a specific protein or an array of different proteins a sample.
2 tahap utama protein assay :
1. Ekstraksi 2. Kuantifikasi ekstraksi protein
Suatu metode purifikasi/pengambilan protein murni dari suatu jaringan
yang terdiri dari berbagai macam zat
1) efficient extraction from biological material
2) separation from non-protein components (nucleic acids and lipids) 3) precipitation steps, initially to recover the bulk protein from a crude extract, followed by preliminary resolution into manageable fraction kuantifikasi protein
Metode “menghitung” protein spesifik/total di dalam suatu jaringan
The aromatic side chains of amino acids absorb light in the UV
range(280 nm). This means that UV absorption can serve as a readout of aromatic amino acid proportion and can help with protein identification and concentration. This is a relatively nonspecific method of quantifying protein concentration. 2. Biuret method
• Protein-copper chelation and secondary detection of reduced copper
• The assay is based on polypeptide chelation of cupric ion (colored chelate) in strong alkali. Compounds containing two or more peptide bonds react with cupric ions (Cu2+) in alkaline solution to produce a complex of reddish-purple colour. • Peptide bond + [Cu2+] -> Cu+ (reddish-purple colour)
a. Lowry method -> Tyr
b. BCA ( Bis-Cinchionic Acid ) method -> AAA (Aromatic Amino Acid) 3. Colorimetric dye based methods
• Protein-dye binding and direct detection of the color change
a. Bradford method Binding of Coomassie dye by protein(AAA) -> Brilliant Blue Easy to use More sensitive Less specific to protein 4. Amino acid analysis
• The use of quantitative total amino acid analysis in the
characterization of protein samples is often overlooked. If you have the possibility to do this, it is the most accurate method for to determine the amount of protein in a sample. • Steps : • Boil in Hcl 120 • Label with proper dye ( Dabsyl-Cl ) • Perform RP-HPLC 5. Fluorescent dye methods
• Protein-dye binding and direct detection of increase in fluorescence
associated with the bound dye
• EZQ fluorescent assay
• Qubit Protein Assay Selecting a protein assay
• The choice among available protein assays is usually based on the
compatibility of the protein assay method with the samples
• Important criteria for choosing an assay include:
• Compatibility with the sample type and components • Assay range and required sample volume • Protein-to-protein uniformity (see below) • Speed and convenience for the number of samples to be tested • Availability of spectrophotometer or plate reader necessary to measure the color produced (absorbance) by the assay PRAKTIKUM Alat & Bahan
Alat : Bahan : Boks stereofoam+es Micropippet Sampel (hepar, otot, usus) Eppendorf Lysis buffer Sonicator Homogenizer Reagen S Sentrifugator Reagen A Microplate Bovine serum albumin Spektofotometer Langkah kerja Ekstraksi Protein
1. Siapkan boks stereofoam dan isilah dengan es minimal setengah
dari tinggi boks 2. Timbang sampel kurang lebih 100 mg, masukkan ke tabung 2 ml 3. Tambahkan 1 ml larutan lysis buffer dan taruh ke dalam boks stereofoam 4. Lisiskan sel menggunakan : a. Sonicator selama 30 detik dan taruh kembali kedalam es selama 10 detik b. Homogenizer selama 20 detik, 3000 rpm dan taruh kembali kedalam es selama 10 detik 5. Ulangi langkah no.4 sampai sel-sel lisis sempurna 6. Pindahkan ke dalam tabung Eppendorf 1,5 ml yang baru 7. Sentrifugasi sampel dengan kecepatan 11000 g selama 20 menit pada suhu 18-20 8. Pindahkan supernatan kedalam tabung Eppendorf baru dan taruh ke dalam boks stereofoam Langkah kerja Kuantifikasi Protein(Lowry)
1. Tambahkan 20uL reagen S ke dalam 1mL reagen A dan campur dengan
seksama dengan menggunakan vortex 2. Buat protein standar (2mg/mL bovine serum albumin) menjadi 0, 0,125 , 0,25 , 0,5 , 1 dan 2 mg/mL dengan menambahkan larutan buffer yang sama 3. Masukkan 5 uL protein standar dan sampel ke dalam sumuran microplate dan setiap sampel minimal dibuat triplets (tiap 1 sampel menggunakan 1 tip) 4. Tambahkan 25 uL reagen pada langkah 1 ke dalam tiap sumuran 5. Kemudian tambahkan 200 uL reagen B ke dalam setiap sumuran 6. Masukkan microplate ke dalam spektrofotometer dan campur dengan seksama selama 5 detik 7. Inkubasi selama 15 menit dan baca nilai absorbansi pada Panjang gelombang 650-750 nm Daftar pustaka
