Metode Bradford didasarkan pada pengikatan secara langsung zat warna Coomassine Brilliant Blue G250 (CBBG) oleh protein yang mengandung residu asam amino dengan rantai samping aromatik (Tyrosine, Tryptophan dan Phenylalanine) atau bersifat basa (Arginine, Histidine dan Leucine). Reagen CBBG bebas berwarna merah-kecoklatan (λmaks 465 nm), sedangkan dalam suasana asam reagen CBBG akan berada dalam bentuk anion yang akan mengikat protein membentuk warna biru (λmaks 595 nm). Jumlah CBBG yang terikat pada protein proporsional dengan muatan positif yang ditemukan pada protein.

Reagen:

    Coomassie Blue G250; sebanyak 100 mg Coomassie Blue G250 dilarutkan dalam 50 mL etanol 95%. Larutan ini kemudian dicampur dengan 100 mL asam fosfat 85%, diencerkan hingga 1 L dengan akuades. Reagen kemudian disaring dengan kertas Whatman No. 1 sebelum disimpan pada suhu kamar. Reagen ini stabil untuk beberapa minggu, meskipun akan terjadi sedikit pengendapan.

Larutan standard:

    Buat seri kadar larutan standard BSA (Bovine Saline Albumin) 0.1 – 1.0 mg/mL dengan pengenceran dari larutan stok.

Prosedur:

  1. Ambil 100 μL larutan (sampel/standard), masukkan ke tabung.
  2. Tambahkan 5 mL reagen, homogenkan. Hindari terjadinya gelembung (busa).
  3. Ukur absorbansi pada 595 nm terhadap blanko (larutan PBS).

Interferensi:

    Detergent: Brij, Triton X, Tween, SDS, CHAPS, CHAPSO, MEGA 10, Octyl-b-D-glucoside
    Solven organik: ethanol, isopropanol, DMSO
    Agen pengkhelat: EDTA, DTPA
    Garam: NaCl, KCl, Na-asetat, NaHCO3, Na-deoxylate
    Buffer: Sitrat pH 5.0, MES pH6.1, Tris pH 7.4, HEPES pH 7.5, CHES pH 9.0
    Agen pereduksi: glukosa, glutatione, asam askorbat, dithiothreitol (DTT), 2-mercaptoethanol

Catatan:

    Reagen CBBG berinteraksi secara kuat dengan kuvet kuarsa. Oleh karena itu, gunakan kuvet gelas atau platik.

Referensi:

    Bradford, M.M., 1976, A Rapid and Sensitive Method for the Quantification of Microgram Quantities of Protein Utilizing the Principle of Protein-dye Binding, Anal. Biochem., 72: 248-254 Dennison C., 2002, A Guide to Protein Isolation, Kluwer Academic Publishers, New YorkHammond, J.B.W. & Kruger, N.J., The Bradford Method for Protein Quantification, In: New Protein Techniques, Methods of Molecular Biology Volume 3, Humana Press, Totowa, New Jersey.

    Olson, B.J.S.C., & Markwell, J., 2007, Assay for Determination of Protein Concentration, In: Current Protocols in Protein Science, John Wiley & Sons, New York.

IN ENGLISH

Bradford method is based on direct binding dyes Coomassine Brilliant Blue G250 (CBBG) by proteins that contain amino acids residues with aromatic side chains (tyrosine, Tryptophan and Phenylalanine) is alkaline or (Arginine, histidine and Leucine). Reagents CBBG free red-brown (λmaks 465 nm), whereas in acid reagent CBBG will be in the form of an anion which will form the blue binding protein (λmaks 595 nm). Total CBBG bound to proteins is proportional to the positive charge found in proteins.

Reagents:

Coomassie Blue G250; as much as 100 mg of Coomassie Blue G250 dissolved in 50 mL of 95% ethanol. This solution is then mixed with 100 mL 85% phosphoric acid, diluted to 1 L with distilled water. Reagent is then filtered with Whatman paper No. 1 before storage at room temperature. This reagent is stable for several weeks, though little precipitation will occur.

Standard solution:

Create a series of standard solution concentration BSA (Bovine Albumin Saline) 0.1 – 1.0 mg / mL by dilution from stock solution.

Procedure:

1. Take 100 μL of solution (sample / standard), enter into the tube.
2. Add 5 mL of reagent, homogenkan. Avoid the bubbles (foam).
3. Measure absorbance at 595 nm against blank (PBS solution).

Interference:

Detergent: Brij, Triton X, Tween, SDS, chaps, CHAPSO, MEGA 10, Octyl-BD-glucoside
Organic solvents: ethanol, isopropanol, DMSO
Chelating agents: EDTA, DTPA
Salts: NaCl, KCl, Na-acetate, NaHCO3, Na-deoxylate
Buffer: Citrate pH of 5.0, MES pH6.1, Tris pH 7.4, HEPES pH 7.5, CHES pH 9.0
Reducing agent: glucose, glutatione, ascorbic acid, dithiothreitol (DTT), 2-mercaptoethanol

Note:

Reagents CBBG kuvet interact strongly with the quartz. Therefore, use glass or platik kuvet.

Reference:

Bradford, MM, 1976, A Rapid and Sensitive Method for the Quantification of Microgram Quantities of Protein Utilizing the Principle of Protein-dye Binding, Anal. Biochem., 72: 248-254

Dennison C., 2002, A Guide to Protein Isolation, Kluwer Academic Publishers, New York

Hammond, J.B.W. & Kruger, NJ, The Bradford Method for Protein Quantification, In: New Protein Techniques, Methods of Molecular Biology, Volume 3, Humana Press, Totowa, New Jersey.

Olson, BJSC, & Markwell, J., 2007, Assay for Determination of Protein Concentration, In: Current Protocols in Protein Science, John Wiley & Sons, New York.

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 ....

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s