3. makronutrian PROTEIN ANALYSIS fix.pdf

analisis protein

1. MK ANALISIS MAKANAN MINUMAN
2025
PROTEIN ANALYSIS

2. PROTEIN
Protein adalah nutrisi kompleks yang
memiliko molekul nitrogen, yang tersusun
dari asam amino dengan ikatan peptida.
Protein memiliki gugus amina (NH2) yang
membedakannya dengan karbohidrat atau
lipid.
Protein disintesis pada jaringan hewan
maupun tumbuhan

3. STRUKTUR UMUM PROTEIN
Sifat Amfoter
dengan asam dan
basa akan
membentuk garam
yang dapat
mengalami ionisasi
Gugus -NH2 bebas
melekat pada
ujung kiri dan
gugus
COOH bebas
melekat pada
ujung kanan
molekul

4. STRUKTUR MOLEKUL PROTEIN
Protein terdiri dari asam amino-asam amino, terikat
bersama-sama dalam bentuk ikatan peptida (-CO-
NH-) melalui proses kondensasi.
Pada pembentukan ikatan peptida, air dilepaskan
dari H pada gugus a-NH2 suatu asam amino dan
dari OH pada gugus a-COOH asam amino yang
lain

5. 1
2
3
FUNGSI PROTEIN
Essential for growth and tissue care
Essential compound precursor (enzyme,
hormone, hemoglobin, neurotransmitter
Control body liquid balance (intracellular
liquid, extracellular liquid and
intravascular liquid)
4
Maintain accumulation of acid/base
5
Stimulate antibody production
6
Nutrient transporter (carrier protein)
7
Energy source (4kkal/gr)

6. Klasifikasi Jenis
Source
Non essential protein Ala, Gln, Glu, Asp, Asn
Essential protein Leu, Ile, Val, Trp, Phe, Thr, Lis, His, Met
Conditional Essential Pro, Ser, Arg, Cys, Gly, Tyr
Amino Acid Precursor
Met, Ser : Cys ; Phe : Tyr ; Glu, Gln, Asp : Arg ; Glu : Pro
Ser : Gly
Synthesize
Eksogen protein diet
Endogen protein Body tissue
Amino Acid Function
Incomplete protein
Zein (jagung)
Complete protein Glisin (soy), glutenin (wheat), animal prot
Partially complete
Gliadin(wheat)legumin(beans)

7. STRUKTUR PROTEIN

8. TUJUAN ANALISIS PROTEIN PADA SAMPEL
MAK-MIN
Protein analysis in food and drinks is essential for ensuring nutritional quality,
economic value, safety, and accurate labeling.
Nutritional Assessment and Health
Determines the quantity and quality of protein, which is vital for human
health, growth, muscle maintenance, and disease prevention (e.g.,
sarcopenia).
It helps assess amino acid profiles and digestibility, ensuring foods meet
dietary requirements
Quality Control and Labeling
Accurate protein measurement is crucial for food labeling,
supporting regulatory compliance and consumer trust.
It ensures that products meet declared nutritional values,
especially in protein supplements and plant-based drinks
Economic and
Industrial Value
Protein content often determines the economic
value of foods like milk, wheat, and protein
powders.
It impacts pricing, product development, and the
feasibility of new protein sources (e.g., plant-
based or alternative proteins)
Functional and Technological Properties
Supports the evaluation of functional properties such as
solubility, emulsification, foaming, and gelation, which affect
food texture and processing.
It guides the selection and optimization of proteins for
specific food applications
Safety and Authenticity
Analysis can detect protein oxidation, adulteration,
or contamination, ensuring food safety and
authenticity

9. Protein Denaturation
is a process in which proteins lose the
tertiary structure and secondary structure
then present in their native state,
by application of some external stress or
compound such as a strong acid or base,
a concentrated inorganic salt, an organic
solvent (e.g., alcohol or chloroform), or
heat
Increase the protein digestibility

10. ISOLASI & PEMURNIAN PROTEIN
Bertujuan untuk memisahkan protein dari material yang tidak diinginkan.
Tahap pertama bertujuan untuk mengisolasi protein dalam bentuk larutan
menggunakan homogenisasi/osmolisis, sehingga protein terpisah dengan sel
atau jaringan.
Metode homogenisasi dilakukan menggunakan blender dengan kecepatan
10.000-15.000 rpm.
Metode osmolisis dilakukan dengan menggunakan larutan hipotonik, (seperti
air atau buffer bebas sukrosa) yang dapat menembus sitosol, sehingga sel
membengkak dan lisis.
Teknik yang digunakan untuk isolasi dan purifikasi didasarkan pada kelarutan
protein, berat molekul, muatan, dan / atau afinitas ikatan protein spesifik.

11. ISOLASI & PEMURNIAN PROTEIN
Extraction of Proteins
Deproteination Procedures
Chemical Deproteination
Deproteination by Thermal
Treatment
Filtration
Ultrafltration Centrifugation
Density Gradient
Centrifugation
Salting-Out
Dialysis and Electrodialysis
Purification and Group
Fractionation by Trap Columns
and Chromatography

12. JENIS PROTEIN
Sarcoplasmic proteins
Soluble in solutions of low salt concentration.
They are most often extracted with phosphoric buffer of ionic strength 0.05 (e.g., 15.5 mM Na2HPO4 13.38 mM KH2PO4, pH 7.3).
As these proteins have been sometimes extracted with pure water, they are commonly known as water-soluble proteins.
Connective tissue
proteins
Composed mostly of collagen and elastin.
These proteins are soluble neither in neutral salt solutions of ionic strength 0.5 nor in 0.05 N solutions of NaOH and HCl; they are
known as stromal proteins.
Myofibrillar proteins
Soluble in solutions of neutral salts of ionic strength 0.5 (in practice, between 0.7 and 1.5) and often known as salt-soluble proteins.
Generally, the 5% NaCl solution buffered to pH 7.0–7.5 (optimally 7.3) by using 0.02–0.003 NaHCO3 is regarded as the best
solvent.
To model conditions of myofibrillar protein extraction for industrial operations (e.g., mincing during preparation of sausage
emulsion), lower NaCl concentra-tions (2.5%–3.0%) are recommended (Kolakowski and Szybowicz 1976).
EXTRACTION OF PROTEIN

13. PROSEDUR DEPROTEINASI
Deproteinasi merupakan prosedur dasar dalam analisis produk makanan.
Deproteinasi bertujuan untuk memisahkan fraksi “nonprotein” dengan “protein”.
Metode deproteinasi melibatkan :
chemical precipitation,
precipitation by thermal treatment,
ultrafltration,
dialysis, or
purification by column chromatography.

14. CHEMICAL PRECIPITATION
Organic solvents (such as ethanol, acetone, and acetonitrile) affect the spatial structure of
a protein molecule, weaken its hydrophobic interactions, and directly react with charged
groups on the protein molecule's surface.
This damages the water coat of the molecule and leads to protein denaturation.
The complete denaturation, occurs only at high concentrations of the precipitating agents
(65% –80%), at an elevated temperature (20°C–30°C), and after an extended time of
precipitation (24 hours).
Acetone with 1% HCl brings about complete deproteination as early as after 15-30
minutes; precipitation with pure acetone requires a longer time, usually from 3 hours to
overnight.

15. Deproteination by Thermal Treatment
A protein denatured thermally at an isoelectric point is nonsoluble.
Acidifcation of water, used as a heating medium, to the isoelectric point of the protein
present in the sample greatly facilitates and accelerates deproteination.
Acetic acid (0.12% final concentration) is used most frequently.
Proteins differ in the temperature of their thermal denaturation; the process may start
within a temperature range of 80°C–100°C and is completed after 3-5 minutes of heating.

16. Speed
Times
(minutes)
the nuclei, intact cells, connective tissue,
sarcolemmal sheaths, and myofbrils
200 × g / 1336 rpm 10–15
Sarcosomes
2,000–10,000 × g /
4226 - 9449 rpm.
10-20
Lysosomes and "heavy" microsomes
10,000–25,000 × g /
9449 - 14940 rpm
20-30
Microsomes, polysomes, ribosomes, and
sarcolemma fragments
25,000–100,000 × g /
14940 – 29881 rpm
120-180
CENTRIFUGATION
Effciency of centrifugation in removing dispersed particles and suspensions from tissue protein extracts depends
primarily on the g value, centrifugation time, and density of particles.

17. method of analysis
2,4,6-Trinitrobenzene
1-Sulfonic Acid
Spectrophotometric Method
Kjeldahl Method
Ultraviolet Absorption
2,4,6-Trinitrobenzene 1-Sulfonic Acid-Spectrophotometric Method
Biuret Method and Its Modifcations
Classic Procedure
Colorimetric Determination of Protein in the Presence of Thiols
Colorimetric Determination of Protein in the Presence of TCA-Soluble Interference Substances
Colorimetric Determination of Protein in Lipid-Rich Samples
Colorimetric Determination of Protein in the Presence of Reducing Saccharides
Spectrophotometric Determination of Protein in the Presence of Deoxyribonucleic Acid
The Pope and Stevens Method
The Classic Pope and Stevens Procedure
Spectrophotometric Modifcation of the Pope and Stevens Method
Formol Titration
Alcohol Titration
Lowry Method
Bicinchoninic Method
Dye-Binding Methods
Ninhydrin Method
Hydroxyproline Determination
N-Amide Determination

18. method of analysis
2,4,6-Trinitrobenzene
1-Sulfonic Acid
Spectrophotometric Method
Chromatography
Filter Paper Chromatography
Thin-Layer Chromatography
Gel-Filtration Column Chromatography
Ligand-Exchange Chromatography on Cu2+-Sephadex
Ion-Exchange Chromatography
High-Performance Liquid Chromatography
Affinity Chromatography
Gas Chromatography
•Electrophoresis
SDS-Polyacrylamide Gel Electrophoresis
Isoelectric Focusing in Polyacrylamide Gel
Two-Dimensional Electrophoresis
Capillary Electrophoresis
Immunochemical Methods
Enzyme-Linked Immunosorbent Assay
Western Blotting

19. DIRECT & UNDIRECT METHOD
2,4,6-Trinitrobenzene
1-Sulfonic Acid
Spectrophotometric Method
Direct Methods
Direct methods measure protein content by quantifying the actual amino acids or protein molecules present.
Examples:
Amino Acid Analysis: Proteins are hydrolyzed into amino acids, which are then quantified, typically by chromatography.
This is considered the most accurate method, as it is not affected by non-protein nitrogen or interfering substances, but
it is time-consuming and costly.
Direct Kjeldahl Analysis: Protein is first isolated and then its nitrogen content is measured, providing a more accurate result than
indirect Kjeldahl.
Direct Mass Spectrometry: Used for identifying and quantifying proteins or modifications directly from samples.
Direct Immunoassays (e.g., direct ELISA): Detects proteins using antibodies that bind directly to the target protein.
Indirect Methods
Indirect methods estimate protein content by measuring a related property (often nitrogen content) and converting it to protein using a
factor.
Examples:
Kjeldahl and Dumas Methods: Measure total nitrogen in a sample and multiply by a conversion factor (commonly 6.25) to estimate
protein. These methods can overestimate protein due to non-protein nitrogen.
Colorimetric Assays (e.g., Bradford, Lowry, Biuret): Rely on chemical reactions with protein functional groups, but can be influenced
by other substances in the sample.
Indirect Immunoassays (e.g., indirect ELISA): Use a secondary antibody for detection, increasing sensitivity but adding complexity.

20. Metode Kjeldahl
2,4,6-Trinitrobenzene
1-Sulfonic Acid
Spectrophotometric Method
Analisis protein kuantitatif protein tak langsung
Penentuan protein kasar (crudeprotein)
Pengukuran kadar Nitrogen dalam bahan pangan
Tahapan : destruksi, destilasi, titrasi
Kadar Protein = Kadar Nitrogen x faktorkonversi
Faktor konversi : 100/16 = 6.25 (umum)
Adavantages Disadvantages
1.Applicable to all types of foods
2.Inexpensive (if not using an automated
system)
3.Accurate; an official method for crude
protein content
4.Has been modified (micro Kjeldahl
method) to measure microgram
quantities of protein)
1.Measures total organic nitrogen, not
just protein nitrogen
2.Time consuming (at least 2 hr to
complete)
3.Corrosive reagent
4.Poorer precision than the Biuret
method
Senyawa lain selain protein yang
mengandung N terukur sebagai protein
(urea, amonia, asam nukleat, nitrit,
nitrat, amida, purin, pirimidin)

21. Metode Kjeldahl
Tahap Destruksi
Tujuan : melepaskan nitrogen dari protein
Sampel dipanaskan dalam larutan asam sulfat pekat
Unsur C dan H teroksidasi menjadi H2O, CO2, CO
Unsur N berubah menjadi amonium sulfat (NH4)2SO4
Asam sulfat juga mendestruksi KH dan lemak
Diperlukan katalisator untuk mempercepat proses destruksi
Tujuan: Mempertinggi titik didih asam sulfat, Suhu destruksi lebih tinggi
(370-410C)
Campuran Na2SO4 dan HgO(20:1)
K2SO4
CuSO4
Tahap Destilasi
Dilakukan dengan menambahkan NaOH
Pada tahap ini amonium sulfat dipecah menjadi amonia
Amonia yang dibebaskan ditampung dalam larutan asam standar biasanya
HCl atau asam borat 4% yang jumlahnya berlebihan
. Tahap titrasi
Jika larutan asam penampung yang digunakan HCl, sisa asam klorida
yang tidak bereaksi dengan amonia (membentuk NH4Cl) dititrasi dengan
NaOH
Jika digunakan indikator PP, TAT larutan menjadi merah muda permanen
(dari asam ke basa)/jika digunakan indikator MR larutan berubah menjadi
kuning
Buat titrasi untuk blanko (tanpa sampel)

22. Metode Kjeldahl
Tahap titrasi
Jika larutan penampung adalah asam borat/HBO3 (asam lemah), banyaknya
asam borat yang bereaksi dengan amonia dapat diketahui dengan titrasi
dengan HCl 0.1N dengan indikator MR+BCG
HCl akan mentitrasi amonium-borat menjadi amonium klorida sehingga pada
akhir titrasi terjadi kelebihan HCl/asam kuat
Akhir titrasi ditandai dengan perubahan larutan dari biru/hijau menjadi merah
muda
Perhitungan
Reaction : H2BO3– + H+ ® H3BO3
Calculation
Moles of HCl = moles of NH3 = moles N in the sample
% N= ml NaOH (blanko-sampel) X N NaOH X 14.008 X 100%
berat sampel (g) X 1000
Kadar protein : % N x faktor konversi

23. BIURET METHOD
Pengukuran jumlah ikatan peptida dalam protein
Semakin tinggi kadar protein bahan, jumlah ikatan peptida semakin
banyak
Prinsip analisis: bahan yang mengandung ikatan peptida dua atau
lebih membentuk kompleks berwarna ungu dengan ion Cu2+/kupri
pada kondisi alkali
The absorbance of the color produced is read at 540 nm
The color intensity (absorbance) is proportional to the protein
content of the sample
Hasil yang diperoleh dapat dipengaruhi oleh adanya
lipida dan komponen lain yang dapat mengubah warna
ataupun memberikan respons yang sama dengan ikatan
peptida.
Contoh gugus yang memberi respons yang sama
dengan ikatan peptida :
–CSNH2 –CHNH2CH2OH
–C(NH)NH2 –CHNH2CHOH
–CH2NH2 –CHOHCH2NH2
–CRHNH2

24. BIURET METHOD
Adavantages Disadvantages
Lebih murah daripada metoda Kjeldahl,
cepat (dapat selesai dalam < 30 menit),
paling sederhana
Deviasi warna jarang terjadi dibanding
dengan metoda Lowry, absorpsi UV,
atau turbidimetri
Senyawa yang dapat mengganggu
analisis hanya sedikit sekali
Tidak mendeteksi nitrogen dari sumber
nonprotein
Kurang sensitif bila dibandingkan
dengan metoda Lowry; perlu minimal
2 – 4 mg protein untuk analisa
Absorbansi dapat berasal dari
pigmen empedu, bila pigmen
tersebut ada di dalam sampel
Garam amonium dengan
konsentrasi tinggi dapat
mengganggu reaksi
Warna yang dihasilkan bervariasi
dengan perbedaan jenis protein.
Misal : gelatin menghasilkan warna
pinkish-purple
Kondisi “tak tembus cahaya” dapat
terjadi pada larutan final apabila
terdapat lipida/karbohidrat dalam
jumlah banyak
Bukan merupakan metoda yang
absolut : warna harus
distandardisasi dengan protein yang
diketahui (misal BSA) atau
dicocokkan dengan metoda Kjeldahl
Pembuatan kurva standar
Buat larutan standar BSA atau kasein dalam air dengan konsentrasi
0.5 mg/ml.
Masukkan ke dalam tabung reaksi 0 (blanko), 0.1, 0.2, 0.4, 0.6, 0.8.
dan 1.0 ml larutan protein standar. Tambahkan air sampai volume total
masing2 sebanyak 4 ml.
Tambahkan 6 ml pereaksi Biuret ke dalam masing2 tabung reaksi.
Campur rata.
Simpan tabung pada suhu 37°C selama 10 menit atau pada suhu
kamar selama 30 menit sampai terbentuk warna ungu yang
sempurna.
Ukur absorbansi pada panjang gelombang 520 nm.
Preparasi sampel
Timbang sampel padat. Hancurkan sampel padat dengan
menggunakan waring blender. Hancuran yang diperoleh disaring lalu
disentrifugasi.
Supernatandidekantasi untuk dipergunakan selanjutnya (protein yang
terdapat dalam supernatan adalah soluble protein).
Sampel cair yang berupa protein konsentrat, isolat yang tidak keruh,
maka persiapan sampel cukup dengan pengenceran saja. Jika
cairannya keruh atau mengandung bahan-bahan yang menganggu
seperti glukosa maka harus dilakukan perlakuan sebagai berikut:

25. BIURET METHOD
Preparasi Sampel (Ekstrak)
Timbang ekstrak. Ekstrak dimasukkan ke dalam tabung
reaksi seperti pada waktu penetapan standar, kemudian
tambahkan air sampai volume total masing-masing 1ml.
Tambahkan 1 ml TCA (Tri Chloroacetic Acid) 10% pada
masing-masing tabung reaksi sehingga protein
akanterdenaturasi.
Sentrifusa pada 3000 rpm selama 10 menit sampai
protein yang terdenaturasi mengendap, supernatan
dibuang dengan caradekantasi.
Ke dalam endapan tambahkan 2 ml etil eter, campur
merata lalu sentrifusa kembali untuk menghilangkan
residu TCA. Biarkan mengering pada suhu kamar.
Ke dalam endapan kering ditambahkan air 4 ml, campur
merata.
Tambahkan 6 ml pereaksi biuret, alkali dalam pereaksi ini
akan melarutkan endapan yang tersisa
0.1-1.0 ml sampel (dipipet tepat) dimasukkan ke dalam
tabung reaksi, kemudian diperlakukan seperti penetapan
standar
Hubungan antara absorbansi pada 540 nm pada Biuret
method dengan kadar protein menurut metoda Kjeldahl

26. LOWRY METHOD
Principle
The Lowry method combines the biuret reaction with the
reduction of the Folin-Ciocalteau phenol reagent
(phosphomolybdic-phosphotungstic acid) by tyrosin &
tryptophan residues in the protein
The bluish color developed is read at 750 nm (high
sensitivity for low protein concentration) or 500 nm (low
sensitivity for high protein concentration)
Prosedur
Terdapat 2 macam reagen Lowry:
Lowry A (fosfotungstat-fosfo molibdat 1 : 1)
Lowry B (Na-karbonat 2% dalam NaOH 0,1N serta
Cu-sulfat dan Na-K-tartrat 2%)
Cara (a):
1 mL larutan protein sampel + 5mL reagen Lowry B,
gojog dan biarkan 10 menit
tambahkan 0,5 mL reagen Lowry A dan biarkan 20
menit
Baca nilai absorbansi pada 600 nm
Protein reaction with cupric ion under alkaline condition

27. LOWRY METHOD
Cara (b)
Proteins to be analyzed are diluted to an appropriate
range (20 – 100 mg).
K Na Tartrate-Na2CO3 solution is added after cooling and
incubated at room temperature for 10 min.
CuSO4-K Na Tartrate-NaOH solution is added after
cooling and incubated at room temperature for 10 min.
Freshly prepared Folin reagent is added, then the
reaction mixture is mixedand incubated at 50oC for 10
min
Absorbance is read at 650 nm
A standard curve of bovine serum albumin (BSA) is
carefully constructed for estimating protein concentration
of the unknown
Cu++ in alkaline solution to form complexity with
protein.
Cu++ catalyses oxidation of phenol group of tyrosine
with phosphomolybdic-phosphotungstic acid.

28. LOWRY METHOD
Adavantages Disadvantages
Very sensitive :
50 – 100 times more sensitive than biuret method
10 – 20 times more sensitive than 280nm UV
absorption method
Similar sensitivity as Nesslerization; however, more
convenient than Nesslerization
Less affected by turbidity of the sample
More specific than most other methods
Relatively simple, can be done in 1 – 1.5 hr
Color varies with different proteins to a greater
extent than biuret method
Color is not strictly proportional to protein
concentration
The reaction is interfered with to varying degrees
by sucrose, lipids, phosphate buffers,
monosaccharides, and hexoamines
High concentration of reducing sugars, ammonium
sulfate, and sulfhydryl compounds interfere with
the reaction

29. TITRASI FORMOL
Prinsip
Larutan protein dinetralkan dengan basa (NaOH),
kemudian ditambah formalin sehingga terbentuk dimetilol
Dengan terbentuknya dimetilol maka gugus amino protein
sudah terikat dan tidak mempengaruhi reaksi antara
asam (gugus karboksil) dengan basa NaOH sehingga
akhir titrasi dapat diakhiri dengan tepat
Indikator yang dipakai adalah PP
Akhir titrasi ditandai saat terjadinya perubahan warna
menjadi merah muda yang tidak hilang dalam 30 detik
Titrasi formol baik untuk digunakan untuk evaluasi proses
terjadinya pemecahan protein (misal : pada fermentasi
protein pada tempe, kecap, tauco, dzsb.)
Proses hidrolisis protein ditandai dengan meningkatnya
titrasi formol.
Protein reaction with cupric ion under alkaline condition

30. TITRASI FORMOL
Prosedur
10 ml larutan protein sampel + 20 ml aquades + 0,4 ml
larutan K-oksalat jenuh (K-oksalat : air = 1 : 3) dan 1 ml
indikator PP 1%. Diamkan selama 2 menit
Titrasi larutan tersebut dengan 0,1N NaOH sampai
terbentuk warna pink atau warna standar (10 ml susu +
10 ml aquades + 0,4 ml K-oksalat jenuh + 1 tetes
indikator rosanilin-khlorida 0,01%)
Setelah warna tercapai, tambahkan 2 ml formaldehid
40% dan titrasilah kembali dengan larutan NaOH sampai
warna seperti warna standar tercapai lagi. Catatlah nilai
titrasi kedua ini.
Dibuat titrasi blanko yang terdiri dari : 20 ml aquades +
0,4 ml larutan K-oksalat jenuh + 1 ml indikator PP + 2 ml
formal-dehid, dan titrasilah dengan larutan NaOH
Titrasi formol = titrasi terkoreksi = titrasi kedua – titrasi
blanko
Interpretasi Data
Bila nilai titrasi formol akan digunakan untuk
menentukan kadar protein, maka harus dibuat
percobaan serupa dengan menggunakan larutan
yang telah diketahui kadar proteinnya (misalnya
dengan metoda Kjeldahl)
Selanjutnya ditentukan hubungan antara titrasi
formol dengan % protein.
Misal :
% protein susu = 1,83 x ml titrasi formol
% kasein = 1,63 x ml titrasi formol

31. ULTRAVIOLET (UV) 280 nm METHOD
Prinsip
Protein show strong absorption at 280 nm, primarily due
to tyrosine (Tyr) and tryptophan (Trp) residues in the
protein
Because the content of Tyr & Trp in protein from each
food source is fairly constant, the A280 could be used to
estimate the concentration of protein
Prosedur
Protein are solubilized in buffer or alkali
Absorbance of protein solution is read at 280 nm against
a reagent blank
Protein concentration is calculated according Beer’s law :
A = abc
A = absorbance, a = konstanta (molar absorp- tivity for
individual protein), b = cuvette path length, c =
concentration
Bisa juga memakai standar BSA
Application
It has been used to determine the protein content of
milk and meat products
It has not been used widely in food system
It is better applied in a purified protein system
It is also better applied in proteins that have been
extracted in alkali or denaturing agents such as 8M
urea

32. ULTRAVIOLET (UV) 280 nm METHOD
Adavantages Disadvantages
Rapid and relatively sensitive (at 280 nm, + 100mg
protein is required, several times more sensitive than the
Biuret method)
Nondestructive : sample can be used for other analysis
after protein determination
No interference from ammonium sulfate and other buffer
salts
Nucleic acid also absorb at 280 nm.
The solution must be clear and colorless. Turbidity
due to particulates in the solution will increase
absorbance falsely
A relatively pure system is required to use this
method