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Protein Analysis

- Protein isolates are characterized/analyzed after each step of purification
 Determination of protein concentration
 Determination of number of subunits
 molecular weight determination
 Assessment of biological activity (especially for enzymes)
Protein Content Determination
1. BIURET ASSAY
• Reagent used: strongly alkaline solution containing Cu2+
• Forms a purple colored solution upon complex formation of Cu2+ with peptides with at
least three amino acid residues
• Intensity of purple color is directly proportional to the amount of protein
• Purple complex absorbs at 540 nm.

2. BRADFORD ASSAY
• Reagent used: Coomasie Brilliant Blue G-250
• Dye-binding method
• CBB G-250 binds with the protein through hydrophobic and electrostatic interactions
• Bradford reagent is initially red; Protein-Bradford reagent mixture is blue
• Blue color absorbs at 595 nm
3. LOWRY ASSAY

Summary

Protein sequencing is mainly relying on chemical or enzymatic digestion methods to separate
peptides and detect the amount and composition of amino acid residues. This post will introduce the
principles and experimental steps of protein sequencing.

Definition

Currently, the so-called protein sequencing refers to the detection of proteins’ primary structure,
which contains the number of polypeptide chains in proteins. Polypeptides and proteins can be used
equally in many cases. Amino acid sequence of polypeptides is the biological function of proteins.

-S-CH2-COOH C. 6. And then it should be protected by alkyl reagents from re-oxidation. to determine the position of disulfide bonds. Analyzing the composition and sequence of peptide fragments. Sequencing N-terminal and C-terminal of polypeptide chains Amino acid of polypeptides is divided into two categories: amino-terminal and carboxyl-terminal. Detecting the amino acid composition of polypeptide chains and calculating the molecular ratio of amino acid composition. Determining the position of disulfide bonds in the original polypeptide chain Generally. Polypeptide can be cleaved into several small peptides. And then 2D-electrophoresis technology will be used to separate each peptide fragment. The N-terminal is much more important in the analysis of amino acid sequence of peptide chains than C- terminal. 5. performic acid: -CH2SO3H B. Reduction + oxidation: Mercaptoethanol.mercaptoethanol under the condition of 8mol / L urea or 6mol / L guanidine hydrochloride. And then comparing it with other peptide fragments. Breaking disulfide bonds Several polypeptides chains are linked by disulfide bonds. which are analyzed by other methods.Sequencing steps 1. 3. More than two methods can be used to break peptide samples into two or more sets of peptides or peptide fragments and then separate them. Determining the amino acid sequencing of each peptide 8. 2. Disulfide bonds will be reduced to thiol with excessive & [beta]. Detecting the number of polypeptide in protein molecules The number of polypeptides can be determined by detecting the relationship between the number of moles of amino acid residues and protein molecular weight. Sulfurous acid decomposition: -R1-S-S-R2 + HSO3-. . which is known as oligomeric protein.+ R2-S-SOH3 4. pepsin will be used to deal with those polypeptide chains with disulfide bonds. which may contain disulfide bonds. For example. Determining the sequence of peptide fragments in polypeptide chains 9. Cleaving and protecting disulfide bonds A. Several polypeptides are combined together by non-covalent bond. 7. Splitting polypeptide chain Protein molecules should be separated and purified. R1-S. DTT +iodoacetic acid. 8 mol/L urea or 6mol / L guanidine hydrochloride can be used to deal with tetramer—Hb and dimer—Enolase.

With the chemical method. Long RNA synthesis is generated using bacteriophage polymerase such as T7.What is the difference between chemical synthesis and enzymatic synthesis (IVT)? The Chemical ligation method ensures the exact sequence that is requested to be made. Depending on the complexity and length of the sequence. if the RNA is generated using polymerase T7 and T3. For most molecular biology applications. T3 or SP6. The Enzymatic method (IVT) allows very long sequences (up to 3. The difference to this method is that the sequence could have three added bases leading the sequence as result generated by the promoter. we can make up to 250 bases long. The chemical method also has a very low yield. it could generate an additional GGG and SP6 could generate an additional GAA as a result of promoter sequence. .000 and higher) to be generated and has a much higher yield. and certain modifications and labeling could also be incorporated. three extra bases on the 5' end shouldn't interfere with anything considering the much longer entire RNA. so the overall cost is higher than the alternative method.