techniques involved

1. General techniques used for
identification and characterization of
proteins and nucleic acids

2. Measurement of Protein
Biochemical research often requires quantitative measurement of protein in solutions.
Several techniques have been developed; however, most have limitations
Amino acid content varies from protein to protein, no single assay will be suitable.
Here, we discuss five assays, in most methods chemical reagents are added to protein
solutions to develop a color whose intensity is measured in a spectrophotometer.
A “standard protein” of known concentration is also treated with same reagents & a
calibration curve is constructed.
The other assay relies on direct spectrophotometric measurement.
The various methods are compared in Table 3.3.

4. Biuret Assays
Substances containing two or more peptide bonds react with biuret reagent, alkaline
copper sulfate, a purple complex is formed.
The colored product is the result of coordination of peptide nitrogen atoms with Cu+2.
The amount of product formed depends on the concentration of protein.
Calibration curve must be prepared by using a standard of protein to be assayed.
A relative standard must be selected that provides a similar color yield.
An aqueous solution of bovine serum albumin (BSA) is a commonly used standard.
Measurements of absorbance at 540 nm (A540) are made against a blank.

5. Continue…..
The data are plotted versus protein concentration (mg/mL) or amount of protein (mg).
Unknown protein samples are treated with biuret reagent & the (A540) is measured after
color development.
The protein concentration is determined from the standard curve.
The biuret assay has several advantages, including speed, similar color development
with different proteins, and few interfering substances.
Its primary disadvantage is its lack of sensitivity.

6. Lowry Protein Assay
Principle behind color development is identical to that of biuret assay except that a second
reagent (Folin-Ciocalteu) is added to increase the amount of color development.
Two reactions account for intense blue color that develops through
the coordination of peptide bonds with alkaline copper (biuret reaction),
reduction of Folin-Ciocalteu reagent by tyrosine & tryptophan residues in protein.
The procedure is similar to that of biuret assay.
Obvious advantage of Lowry assay is its sensitivity however, more time is required.
Since proteins have varying contents of tyrosine and tryptophan, the amount of color
development changes with different proteins, including the bovine serum albumin standard.
For this, the Lowry protein assay should be used only for measuring changes in protein
concentration, not absolute values of protein concentration.

7. Bradford Assay
This assay is based on protein binding of a dye, provides numerous advantages over
other methods.
Binding of Coomassie Brilliant Blue dye (Figure 3.5) to protein in acidic solution causes
a shift in wavelengh of maximum absorption (λmax) of dye from 465 nm to 595 nm.
The absorption at 595 nm is directly related to the concentration of protein.
The dye reagent interacts primarily with positively-charged A. A in proteins.
In addition, there are probably weaker interactions between the dye molecules &
hydrophobic, aromatic amino acid residues.
Proteins that have more than an average number of these interacting residues show
higher sensitivity and thus higher absorbance values at 595 nm.

8. Calibration curve is prepared by plotting µg of standard protein versus absorbance at
595 nm, used to estimate amount of protein in unknown samples (Figure 3.6).
Recommended standard proteins include bovine gamma globulin (IgG), lysozyme, &
ovalbumin as closer to average or typical number of A.A. residues that bind the dye.
Although BSA is often recommended in the literature, it is not a good choice.
The 595 nm absorbance of BSA is about 2.1 times that of IgG (Figure 3.6).
This assay requires only a single reagent, i.e. Coomassie Brilliant Blue G-250.
After addition of dye solution to a protein sample, color development is complete in 2
minutes & the color remains stable for up to 1 hour.
Continue…..

9. Sensitivity of Bradford assay rivals & may surpass that of the Lowry assay.
With a microassay procedure, the Bradford assay can be used to determine proteins in
the range of 1 to 20 µg.
Bradford assay shows significant variation with different proteins, but this also occurs
with Lowry assay & can be avoided by use of the proper protein standard.
Bradford method not only is rapid, but also has very few interferences by non-protein
components.
The only known interfering substances are the detergents Triton X-100 & sodium
dodecyl sulfate.
Continue…..

12. BCA Assay
The BCA protein assay is based on chemical principles similar to those of biuret &
Lowry assays.
Protein to be analyzed is reacted with Cu+2 & bicinchoninic acid (BCA).
The Cu+ is chelated by BCA, converts the apple-green color of free BCA to purple color
of copper- BCA complex.
Samples of unknown protein & relative standard are treated with the reagent, the
absorbance is read in a spectrophotometer at 562 nm
Concentrations are determined from a standard calibration curve.
This assay has the same sensitivity level as the Lowry & Bradford assays.
Main advantages are its simplicity & usefulness in the presence of 1% detergents such
as Triton or sodium dodecyl sulfate.

13. Spectrophotometric Assay
Most proteins have relatively intense ultraviolet light absorption centered at 280 nm.
Absorbance taken at A280 gives value of a protein that is proportional to protein conc.
In practice, the procedure is simple and rapid.
A protein solution is transferred to a quartz cuvette & measure its absorbance at A280
against a reference cuvette containing the protein solvent only.
For average, a conc. of 1 mg/mL will yield an absorbance at 280 nm of about 1.
If the extinction coefficient is known for protein, its exact concentration may be
calculated from absorbance at 280 nm.
Cellular extracts contain many other compounds that absorb in the vicinity of 280 nm.
Nucleic acids, which can be common contaminants in a protein extract, absorb strongly
at 280 nm (λmax = 260).

14. Early researchers developed a method to correct for this interference by nucleic acids.
Mixtures of pure protein & pure nucleic acid were prepared, and the ratio was
experimentally determined.
The following empirical equation may be used for protein solutions containing up to 20%
nucleic acids. “Protein conc. (mg/ml) = 1.55A280 - 0.76A260”
Although, this method is fast, relatively sensitive, & requires only a small sample size, it
is still only an estimate of protein conc. unless its extinction coefficient is known.
It has certain advantages over colorimetric assays i.e. most buffers & ammonium sulfate
do not interfere & the procedure is nondestructive.
It is particularly suited for rapid & continuous measurement of protein elution from a
chromatography column, where only protein concentration changes are required.
Continue…..

15. Measurement of Nucleic Acid (N.A)
Spectrophotometric Assay
Solutions of N.A strongly absorb ultraviolet light with a of λmax 260 nm.
Intense absorption is due to the presence of aromatic rings in purine & pyrimidine bases.
Conc. of N.A in solution can be calculated if one the knows value of A260 of solution.
Solution of double stranded DNA at conc. of 50 mg>mL in a 1-cm quartz cuvette will give
a reading of about 1.0.
A solution of single-stranded DNA or RNA that has an A260 reading of 1.0 in a cuvette
with a 1-cm path length has a concentration of about 40 mg>mL
It is simple, rapid, nondestructive, and very sensitive assay; however, it should be noted
that it measures both DNA and RNA.

16. The minimum conc. of N.A that can be accurately determined is 2.5–5.0 mg>mL.
Measuring the ultraviolet absorption of DNA & RNA solutions can also provide a useful
structural probe & an indication of purity.
DNA structural changes such as helix unwinding may be detected by measuring
absorbance changes of DNA solutions at 260 nm.
Ratio of A260/A280 is often used as a relative measure of purity of N.A solutions.
Pure DNA with little or no protein present has an absorbance ratio of about 1.8.
Pure RNA has a ratio about 2.
Absorbance ratios less then these indicate contamination of solution by protein material.
Continue…..

17. Other Assays for N.A
Spectrophotometric assay does not have a sufficient level of sensitivity for many
applications.
Other assays, depend on enhanced fluorescence of dyes when bound to N.A.
Features of fluorescence assays are compared to spectrophotometric assay in Table 3.4
The fluorescence dye bisbenzimidazole, when bound to DNA, emits light at 458 nm that
is about five times greater in intensity than emission by free, unbound dye.
Increase in fluorescence is linear with increasing amounts of DNA in conc. range of 0.01
to 5 µg/ ml.

18. Advantage of this assay is that it can quantify DNA in cell suspensions & crude
homogenate
It does not respond to RNA or other biomolecules in extracts.
The is fast & requires only a fluorimeter.
Continue…..

20. Techniques for Sample
Preparation

21. Dialysis
Oldest procedures applied to the purification & characterization of biomolecules is
dialysis
This involves sealing an aqueous solution containing both macromolecules & small
molecules in a porous membrane.
Sealed membrane is placed in a large container of low-ionic-strength buffer.
The membrane pores are too small to allow diffusion of macromolecules of M.W greater
than about 10,000.
Smaller molecules diffuse freely through the openings (Figure 3.7).
Passage of smaller molecules continues until their conc. inside the dialysis tubing &
outside in large volume of buffer are equal.

22. Thus, the conc. of small molecules inside the membrane is reduced.
Equilibrium is volume dependent & reached after 4 to 6 hours.
If dialysate is replaced with fresh buffer after equilibrium, the conc. of small molecules
inside membrane will be further reduced by continued dialysis.
Dialysis is commonly used to remove salts & other small molecules from solutions of
macromolecules.
During separation & purification of biomolecules, small molecules are added to
selectively precipitate or dissolve the desired molecule.
Dialysis is simple, inexpensive, & effective method for removing small molecules.
Continue…..

23. Also useful for removing small ions & molecules that are weakly bound to biomolecules.
Protein cofactors like NAD, FAD, & metal ions can often be dissociated by dialysis.
Removal of metal ions is facilitated by addition of a chelating agent to dialysate.
Dialysis of small-volume samples is often tedious, inconvenient, & inefficient.
Commercial vials for dialysis are now available for sample sizes from 0.05 to 3.0 mL,
with molecular weight cutoffs ranging from 3500 to 14,000 daltons.
Continue…..

25. Ultrafiltration
Ultrafiltration involves separation of molecular species on the basis of size, shape,
and/or charge.
The solution to be separated is forced through a membrane by an external force.
Membranes may be chosen for optimum flow rate, molecular specificity, & molecular
weight cutoff.
Membrane filtration mostly applied for
desalting buffers or other solutions
clarification of turbid solutions by removal of micron- or submicron-sized particles.
Ultrafiltration membranes have M.W cutoffs in the range of 100 to 1,000,000.

26. They are usually composed of two layers
a thin surface that is semipermeable membrane made from a variety of materials
including cellulose acetate, nylon, & polyvinylidene
a thicker, inert, support base (Figure 3.8)
These filters function by retaining particles on surfaces, not within the base matrix.
Membrane filters of these materials can be manufactured with a predetermined &
accurately controlled pore size.
Filters are available with a mean pore size ranging from 0.025 to 15µm.
Continue…..

27. These filters require suction, pressure, or centrifugal force for liquid flow.
Typical flow rate for commonly used 0.45–µm membrane is 57mL/min-1/cm-2 at 10 psi.
Ultrafiltration devices are available for macroseparations (up to 50 L) or for
microseparations (milli- to microliters).
For solutions larger than a few milliliters, gas-pressurized cells or suction-filter devices
are used.
For concentration & purification of samples in the milli- to microliter range, disposable
filters are available.
These devices, often called microconcentrators, offer user simplicity, time saving, & high
recovery.
Continue…..

28. The sample is placed in a reservoir above the membrane & centrifuged.
The time & centrifugal force required depend on the membrane.
Figure 3.9 outlines the use of a centrifuge microfilter.
The nomenclature implies that separations are result of physical trapping of particles &
molecules by filter.
With polycarbonate & fiberglass filters, separations are made primarily on the basis of
physical size.
Other filters trap particles that cannot pass through the pores, but also retain
macromolecules by adsorption.
Continue…..

29. In particular, these materials have protein & nucleic acid-binding properties.
Each type of membrane displays a different affinity for various molecules.
For protein, the relative binding affinity is polyvinylidene fluoride ˃ cellulose nitrate ˃
cellulose acetate.
Some applications of ultrafiltration are here
Continue…..

31. Clarification of Solutions
Due to low solubility & denaturation, solutions of biomolecules or cellular extracts are
often turbid.
This is a particular disadvantage if spectrophotometric analysis is desired.
Transmittance of turbid solutions can be greatly increased by passage through a
membrane filter system.
This simple technique may also be applied to the sterilization of non-autoclavable
materials such as protein & nucleic acid solutions or heat-labile reagents.
Bacterial contamination can be removed from these solutions by passing them through
filter systems that have been sterilized by autoclaving.

32. Collection of Precipitates for Analysis
Collection of small amounts of very fine precipitates is the basis for many chemical &
biochemical analytical procedures.
Membrane filtration is an ideal method for sample collection.
This is of great advantage in the collection of radioactive precipitates.
Cellulose nitrate and fiberglass filters are often used to collect radioactive samples
because they can be analyzed by direct suspension in an appropriate scintillation
cocktail.

33. Harvesting Bacterial Cells
Collection of bacterial cells from nutrient broths is typically done by batch centrifugation.
This time-consuming operation can be replaced by membrane filtration.
Filtration is faster than centrifugation, & allows extensive cell washing.

34. Concentration of Biomolecule
For concentration of biomolecules, ultrafiltration pressure cells are most effective (Fig
3.10).
A membrane filter is placed in bottom & the solution is poured into the cell.
High pressure, exerted by compressed nitrogen, forces the flow of small molecules,
including solvent, through the filter.
Larger molecules that cannot pass through pores are concentrated in sample chamber.
This method of concentration is rapid & gentle and can be performed at cold
temperatures to ensure minimal inactivation of the molecules.
One major disadvantage is clogging of pores, which reduces the flow rate through the
filter; this is lessened by constant but gentle stirring of solution.

36. Lyophilization
Lyophilization is used for concentration of biological solutions.
It is a drying technique that uses to change a solvent in frozen state directly to vapor
state under vacuum.
The product after lyophilization is a fluffy matrix.
This is the effective methods for drying or concentrating heat-sensitive materials.
In practice, a biological solution to be concentrated is “shell-frozen” on the walls of a
round-bottom or freeze-drying flask.
Freezing of solution is accomplished by placing the flask in a dry ice–acetone bath &
slowly rotating it as it is held at an angle of 45 degree.

37. Aqueous solution freezes in layers on the wall of the flask.
This provides a large surface area for evaporation of water.
Flask is then connected to lyophilizer, which consists of a refrigeration unit & vacuum
pump (Figure 3.11).
Combined unit maintains the sample at -40°C for stability of biological materials &
applies a vacuum of approximately 5 to 25 mm Hg on sample.
Ice formed from aqueous solution sublimes & is pumped from the sample vial.
In fact, all materials that are volatile under these conditions (-40°C, 5 to 25 mm Hg) will
be removed, and nonvolatile materials will be concentrated into a light, sometimes fluffy
precipitate.
Most freeze-dried biological materials are stable for long periods & some remain viable
for many years.
Continue…..

39. Only aqueous solutions should be lyophilized.
Organic solvents lower the melting point of aqueous solutions & increase the chances
that the sample will melt & become denatured during freeze-drying.
There is also the possibility that organic vapors will pass through the cold trap into the
vacuum pump, where they may cause damage.
A new & increasingly popular technique for sample concentration & drying is centrifugal
vacuum concentration.
Used to dry a wide variety of biological samples.
Continue…..

40. The process starts with a sample dissolved in a solvent.
Solvent is evaporated under vacuum in a centrifuge, thus producing a pellet in the
bottom of container.
The most widely used instrument is the SpeedVac, available through Savant (Figure
3.12).
This method is better than freeze-drying because it is faster, it does not require a
prefreezing step
It provides 100% sample recovery
May be used with solvents other than water.
The SpeedVac is usually used for relatively small volumes—1–2-mL samples.
Continue…..

42. Centrifugation
Centrifugation is carried out by spinning a biological sample at a high rate of speed, thus
subjecting it to an intense force.
Most centrifuge techniques fit into one of two categories—preparative or analytical
centrifugation.
A preparative procedure is one that can be applied to the separation or purification of
biological samples by sedimentation.
Analytical procedures are used to measure physical characteristics of biological
samples.

43. Instrumentation for Centrifugation
Basic centrifuge consists of two components, an electric motor with drive shaft to spin
the sample & a rotor to hold tubes or other containers of sample.
A wide variety of centrifuges is available like the low-speed or clinical centrifuge; the
high speed centrifuge, including the “microfuge”; & the ultracentrifuge.
Major characteristics & applications of each type are compared in Table 4.1.
Here we discuss ultracentrifuge in detail.

45. Ultracentrifuge
Most sophisticated centrifuges are the ultracentrifuges.
Spin chamber must be refrigerated & placed under a high vacuum to reduce friction.
Sample in a cell or tube is placed in rotor, which is then driven by an electric motor.
Although it is rare, metal rotors sometimes break into fragments when placed under high
stress.
Rotor chamber on all ultracentrifuges is covered with a protective steel armor plate.
The drive shaft of ultracentrifuge is constructed of a flexible material to accommodate
any “wobble” of rotor due to imbalance of samples.
It is important to counterbalance samples before inserting them in the rotor.

46. The centrifuge i.e. low, medium, & high speed are of value only for preparative work.
Ultracentrifuges can be used both for preparative work & analytical measurements.
preparative models; primarily used for separation & purification of samples
analytical models; designed for performing physical measurements on the sample
during sedimentation.
Two of most versatile models are Beckman Optima MAX & TLX microprocessor-
controlled tabletop ultracentrifuges (Figure 4.10).
With a typical fixed-angle rotor, which holds six 0.2- to 2.2-mL samples, can generate
150,000 rpm & an RCF of up to 1,500,000.
Continue…..

47. Analytical ultracentrifuges have same basic design as preparative models, except that
they are equipped with optical systems to monitor directly the sedimentation of sample
during centrifugation.
For analysis, a sample of nucleic acid or protein (0.1 to 1.0 mL) is sealed in a special
analytical cell & rotated.
Light is directed through the sample parallel to the axis of rotation, & measurements of
absorbance by sample molecules are made.
If sample molecules have no significant absorption bands in the wavelength range, then
optical systems that measure changes in the refractive index may be used.
Continue…..

48. Optical systems aided by computers are capable of relating absorbance changes or
index of refraction changes to the rate of movement of particles in sample.
The optical system actually detects & measures the front edge or moving boundary of
the sedimenting molecules.
These measurements can lead to an analysis of concentration distributions within the
centrifuge cell.
Continue…..

51. Recommended book
Biochemistry Laboratory: Modern Theory and
Techniques
Rodney Boyer (Hope College)
Second Edition
Chapter 3 and 4:General laboratory procedures and
Centrifugation techniques in biochemistry