1.Protein Estimation
Protein determination is necessary to estimate the amount of protein
in the sample, to normalise against the protein concentration
or during purification procedures. Depending on the amount of
sample, accuracy and presence of interfering agents, one needs
to decide on the method to be used. For accurate quantification,
the sample protein is compared with a known amount of a standard
protein which could either be the commonly used bovine
serum albumin (BSA) or it could sometimes be immunoglobulin
G (IgG). The various methods and their specifications are outlined
below:
1.1 Absorbance Assays
The aromatic rings in the protein absorb ultraviolet light at an
absorbance maximum of 280 nm, whereas the peptide bonds
absorb at around 205 nm. The unique absorbance property of
proteins could be used to estimate the level of proteins. These
methods are fairly accuratewith the ranges from 20μg to 3mg for
absorbance at 280 nm, as compared with 1 to 100μg for 205 nm.
The assay is non-destructive as the protein in most cases is not
consumed and can be recovered. Secondary, tertiary and quaternary
structures all affect absorbance; therefore, factors such as
pH, ionic strength, etc can alter the absorbance spectrum. This
assay depends on the presence of amino acids which absorb UV
light (mainly tryptophan, but to a lesser extent also tyrosine).
Small peptides that do not contain such amino acids cannot be
measured easily by UV.
Requirements
- Quartz Cuvettes
- UV-Spectrophotometer

Amino polyols are an important part of synthetically created amino acids. They are highly antibacterial and immunosuppressive and so are used in various antibiotics and antifungal products. This reaction shows one of the step necessary in creating the amino polyol.

Benzoyl chloride is added to the polyol to form a tribenzoate compound. In this reaction, the polyol has several R-O-H groups that act as weak nucleophiles. When the benzoyl-Cl bond breaks upon addition to the pyridine solvent, the benzoyl group acts as an electrophile. With the help of the DMAP (dimethylaminopyridine) catalyst in the reaction, the R-O-H group is deprotonated and the benzoyl group is added to the remaining R-O form the final tribenzoylated product. As seen above, the reaction has a yield of about 90%. The OTBS (t-buytldimethylsiloxy) groups do not participate in this reaction.

There is one hydroxyl group left on the molecule produced. All of the hydroxyl groups would be replaced by benzoyl groups if the reaction was not stopped after three groups had been added. To stop the reaction at this point, three equivalents of benzoyl chloride were used for every polyol.