Pengukuran konsentrasi protein secara akurat menjadi sangat penting peranannya mengingat hasil penetapan ini digunakan dalam perhitungan lain, seperti penentuan aktivitas enzim. Kesalahan dalam penetapan kadar protein akan berbuntut pada kesalahan-kesalahan di perhitungan selanjutnya. Berbagai metode analisis kuantitatif telah ditawarkan dengan kelebihan maupun kelemahan masing-masing. Secara umum, metode analisis kuantitatif protein digolongkan dalam:

  • Spektrofotometri:
    – Spektrofotometri UV
    – Kolorimetri
  • Volumetri:
    – metode Kjeldahl (didasarkan pada analisis Nitrogen)
    – titrasi formol
    Kuantifikasi protein secara kolorimetrik pada dasarnya terbagi dalam 2 golongan, yaitu berdasarkan pengikatan dengan zat warna (dye-binding) dan pembentukan kompleks dengan tembaga (copper-chelating).
    Metode-metode berdasarkan pengikatan dengan zat warna meliputi:
  1. Metode Bradford (Coomassie Blue)
  2. Metode Ninhydrin
  3. Metode Bromocresol Green
  4. Metode Erythrosin-B
  5. Metode 3′,3″,5′,5″-tetrabromophenolphtalein ethyl ester (TBPEE)
  6. Metode Woodward’s Reagent K
    Metode-metode berdasarkan pembentukan kompleks dengan tembaga meliputi:
  1. Metode Biuret
  2. Metode Lowry (Folin-Ciocalteu)
  3. Metode Bicinchoninic Acid (BCA)
  4. Metode FeCl3

Standard Protein

    Pemilihan standard protein merupakan penentu keberhasilan analisis kuantitatif. Bovine Serum Albumin (BSA) adalah protein yang umum digunakan sebagai standard dalam penetapan kadar protein. BSA banyak dipilih karena tingkat kemurniannya yang tinggi dan harganya relatif murah. Standard protein lain yang bisa digunakan adalah Bovine Gamma Globulin (BCG), terutama untuk keperluan kuantifikasi antibodi karena BCG menghasilkan warna yang sangat mirip dengan immunoglobulin G (IgG).

    Larutan induk (stok) standard protein biasanya dibuat dalam konsentrasi 1 – 5 mg/mL, namun tidak menutup kemungkinan dibuat larutan stok dengan konsentrasi yang lebih besar. Misalkan larutan stok standard protein yang ingin dibuat adalah 1 mg/mL sebanyak 100 mL, maka ditimbang 100 mg BSA kemudian dilarutkan dalam 100 mL akuades (sebaiknya bebas CO2). Perlu dicatat, ketika larutan stok dibuat, jangan dikocok, karena akan berakibat denaturasi protein (teramati sebagai buih di permukaan larutan). Untuk membantu pelarutan protein, cukup diaduk perlahan. Simpan larutan stok ini (bila belum digunakan) dalam freezer (atau lebih baik pada suhu -20oC).

Preparasi Sampel

    Sebelum sampel dianalisis, sampel harus dilarutkan terlebih dahulu, biasanya dalam buffer fosfat (PBS = Phosphate Buffer Saline). Proses pelarutan dilakukan dalam keadaan dingin. Bila sampel berupa padatan, jaringan atau sel, diperlukan langkah pendahuluan sebelum dilarutkan berupa penghancuran (diblender). Bila perlu, lakukan pemurnian protein untuk meminimalkan interferensi matriks. Cara paling sederhana adalah dengan mengendapkan protein menggunakan aseton

    Prosedur pengendapan protein:

  1. Tambahkan aseton 100% (yang telah didinginkan dalam es) pada larutan sampel hingga diperoleh konsentrasi akhir aseton 80% (misal: 2 mL larutan sampel + 8 mL aseton) dalam tabung sentrifuge.
  2. Vortex campuran tersebut, kemudian diinkubasi pada -20oC (atau pada freezer) selama 1 jam.
  3. Sentrifugasi pada kecepatan 15.000 rpm selama 20 menit pada 4oC.
  4. Buang supernatan, endapan dilarutkan kembali dengan PBS dengan vortex dan encerkan secara kuantitatif hingga volume tertentu.

IN ENGLISH

An accurate measurement of protein concentration becomes very important considering the results of this determination is used in other calculations, such as enzyme activity determination. Errors in the determination of protein content will bear on the errors in subsequent calculations. Various quantitative methods have strengths and weaknesses are offered with each. In general, the methods of quantitative analysis of proteins are classified into:

Spectrophotometry *:
– UV Spectrophotometry
– Colorimetric

* Volumetric:
– Kjeldahl method (based on nitrogen analysis)
– Formol titration

Colorimetric protein quantification is basically divided into two groups, which is based on the binding of the dyes (dye-binding) and complex formation with copper (copper-chelating).

The methods based on dye binding with include:

1. Bradford method (Coomassie Blue)
2. Ninhydrin method
3. Bromocresol Green method
Fourth. Method-B Erythrosin
5. Method 3 ‘, 3’, 5 ‘, 5 “-tetrabromophenolphtalein ethyl ester (TBPEE)
6. Method of Woodward’s Reagent K

The methods based on complex formation with copper include:

1. Biuret Method
2. Lowry method (Folin-Ciocalteu)
3. Methods Bicinchoninic Acid (BCA)
Fourth. FeCl3 method

Standard Protein

The selection is a critical success standard protein quantitative analysis. Bovine serum albumin (BSA) is a protein commonly used as standard in the determination of protein content. BSA was chosen because a high purity level and the price is relatively cheap. Standards of other proteins that can be used is Bovine Gamma Globulin (BCG), especially for quantification of antibodies because BCG produces a color that is very similar to the immunoglobulin G (IgG).

Mother liquor (stock) is usually made in a standard protein concentration of 1-5 mg / mL, but did not rule made stock solution with a greater concentration. Suppose a protein standard stock solution to be made is one mg / mL of 100 mL, then weighed 100 mg BSA and then dissolved in 100 mL of distilled water (preferably free of CO2). It should be noted, when the stock solution is made, do not shake, because it will result in denaturation of proteins (observed as a froth on the surface of the solution). To help the dissolution of protein, slowly stirring enough. Save This stock solution (if not used) in the freezer (or better at a temperature of-20oC).

Sample Preparation

Before the samples analyzed, the sample must be dissolved first, usually in a phosphate buffer (PBS = Phosphate Buffer Saline). Dissolution process carried out in the cold. When a solid sample, tissue or cell, the necessary preliminary step before the destruction of the dissolved form (blender). If necessary, do the purification of proteins to minimize matrix interference. The simplest way is to precipitate the protein using acetone

Protein precipitation procedure:

1. Add acetone 100% (which has been chilled in ice) in the sample solution to achieve the final concentration of acetone 80% (eg: 2 mL + 8 mL sample solution of acetone) in the tube sentrifuge.
2. Vortex the mixture, then incubated at-20oC (or in the freezer) for 1 hour.
3. Speed centrifugation at 15 000 rpm for 20 minutes at 4oC.
Fourth. Discard the supernatant, the sediment was dissolved again with PBS with the vortex, and dilute quantitatively up to certain volume.

About Khamir_Yeast

Pengajar dan Penulis Independen, Penyayang Kucing, Karakter : Humoris tapi Gampang Tersinggung, Agak temperamental dan cepat emosi, Cepat memaafkan, Lebih suka menganalisa sebelum berkomentar, Bersahabat tapi gak suka dengan orang yang Lebay. Suka makanan yang pedas. Hobi melukis, berenang, dan memasak. Moto Hidup : BATU saja bisa PECAH apalagi MASALAH ....

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