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

Jenis :

1. Spectroscopic procedure
2. Biuret method
3. Colorimetric dye based methods
4. Amino acid analysis
5. Fluorescent dye methods
1. Spectroscopic procedure

• UV absorbance
• Spectrophotometry

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

https://www.thermofisher.com/id/en/home/life-science/protein-biology/pr
otein-biology-learning-center/protein-biology-resource-library/pierce-protei
n-methods/overview-protein-assays.html

https://www.sciencedirect.com/science/article/pii/S0076687909630081?vi
a%3Dihub

https://www.ncbi.nlm.nih.gov/pubmed/19892168
danke schön