Question

What is the general protocol/description for the following:

1) preparation of a lysis buffer

2) mass spectrometry

3) electrophoresis (for proteins)

4) enzyme assay (to test enzyme activity)

Please answer ALL QUESTIONS

Answer 1

A lysis buffer is a buffer solution used for the purpose of breaking open cells to analyze the components of the cell. Lysis buffers invariably have to have a salt so as to regulate the acidic condition and osmolarity. Lysis buffers can be added for both plant and animal cells.

The ideal lysis buffer requires the following:-

1. Buffer: A buffer is need to be added to the create the lysis buffer, else it can disrupt the pH balance of the cell and thus interfering with either the protein structure or other components. Thus to maintain the acidic and alkaline nature of the cell the buffer is required.
2. Salts: A salt when put into the lysis buffer it will establish an ionic strength in the buffer solution, thus making it a more effective buffer.
3. Detergents: Detergents are organic surfactants; detergents are used to separate membrane proteins from membrane because the hydrophobic part of detergent can surround biological membranes and thus isolate membrane proteins from membranes.

Mass spectrometry (MS) is an analytical technique detects ionized chemical species and sorts the ions based on their mass-to-charge ratio. To put it in a simple term a mass spectrum measures the masses within a sample at the ionic level. Mass spectrometry is used in many different fields and is applied to pure samples as well as complex mixtures.

In an ideal Mass Spectrum procedure, a sample, which may be any of the three states or even ionized can be used, will be bombarded with electrons. This can cause the sample's molecules to break into ionic charged fragments. Then with an electric or magnetic field ions are then separated according to their mass-to-charge ratio, typically by accelerating them and subjecting them: ions of the same mass-to-charge ratio will undergo the same amount of deflection.

Following the deflection of the ions, they are detected by a mechanism capable of detecting charged particles, such as an electron multiplier (which is a vacuum-tube structure that multiplies incident charges). The results are then displayed as spectra of the relative abundance of detected ions as a function of the mass-to-charge ratio. The atoms or molecules in the sample can be identified by comparing it to known masses such as that of an entire molecule) to the identified masses.

Protein electrophoresis is a method for examining the proteins present in an extract. Electrophoresis can be performed with a small volume of sample in a number of ways with or without a supporting medium. However the most common methods carried out are Polyacrylamide Gel Electrophoresis or SDS Polyacrylamide Gel Electrophoresis(in short: gel electrophoresis, PAGE, or SDS-electrophoresis).

Polyacrylamide Gel Electrophoresis (PAGE)

Proteins have a nature that they are charged molecules and this helps in isolating them to a certain extent. Acrylamide gel serves as a size-selective sieve during separation to differentiate the size of the protein. Since proteins have a charge they tend to move through a gel in response to an electric field, the smaller molecules travel more rapidly than larger proteins,

In most PAGE applications, the gel is mounted between two buffer chambers, and the only electrical path is through the gel. Usually, the gel has a vertical orientation, and the gel is cast with a comb that generates wells in which the samples are applied. Applying an electrical field across the buffer chambers forces the migration of protein into and through the gel.

Two types of buffer systems can be used:

• Continuous buffer systems — use the same buffer (at constant pH) in the gel, sample, and electrode reservoirs. Samples are loaded into wells, and proteins that are closer to the gel enter first. This provides a uniform separation matrix, but yields fuzzy and unresolved protein bands. Continuous systems are rarely used for protein electrophoresis but commonly used for nucleic acid analysis
• Discontinuous buffer systems — use a gel separated into two sections (a large pore stacking gel on top of a small pore resolving gel, see figure below) and different buffers in the gels and electrode solutions. Proteins migrate quickly through the large pore stacking gel and then are slowed as they enter the small pore resolving gel. The proteins stack on top of one another to form a tight band, which helps improve resolution. Discontinuous buffer systems provide higher resolution than continuous systems, and varying the buffers used in the sample, gel, and electrode chambers creates a variety of discontinuous buffer systems that can be used for a variety of applications

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

To overcome the limitations of native PAGE systems a detergent, sodium dodecyl sulfate (SDS) was introduced into a discontinuous buffer system, creating what has become the most popular form of protein electrophoresis, SDS-PAGE.

When proteins are separated in the presence of SDS and denaturing agents, they become fully denatured and dissociate from each other. In addition, SDS binds non-covalently to proteins in a manner that imparts:

• An overall negative charge on the proteins. Since SDS is negatively charged, it masks the intrinsic charge of the protein it binds
• A similar charge-to-mass ratio for all proteins in a mixture, since SDS binds at a consistent rate of 1.4 g SDS per 1g protein SDS (a stoichiometry of about one SDS molecule per two amino acids)
• A long, rod-like conformation on the proteins instead of a complex tertiary shape

As a result, the rate at which an SDS-coated protein migrates in a gel depends primarily on its size, enabling molecular weight determination.