This document discusses methods for extracting and preparing proteins for proteomics analysis. It describes how to prepare stable buffer solutions to maintain pH during extraction. Protease inhibitors are also important to prevent proteolysis during extraction and purification. Various mechanical and enzymatic cell lysis techniques are covered, such as sonication, French press, and enzymatic lysis of yeast. Considerations for extracting from prokaryotes, eukaryotes, and animal tissues are provided. Centrifugation can be used to separate subcellular fractions after homogenization.

1. PROTEOMICS
Topic:Extraction buffer,protease inhibitors methods
of cell distruption
Presented by
T.Ananthakumar

2. 1.1 Preparation of Buffers for Protein Extraction
• Proteins are extremely heterogeneous biological
macromolecules.
• Their properties can be severely affected by small changes in
hydrogen ion concentration, and thus a stable pH of the
protein environment is necessary.
Theory of buffering
• In order to ensure reproducible experimental results, it is
important to maintain the protein solution at the constant pH.
Buffering: partially neutralized solutions of weak acid or weak
bases are resistant to pH changes on the addition of small
amounts of strong acid or strong base.

3. Cont..
HA (acid) ←→H++A- (incomplete) (1.1)
NaA←→Na++ A- (complete) (1.2)
• The addition of small amounts of strong acid (H+) to
the buffer shifts equilibrium (Equation 1.1) to the left
using A- supplied by Equation 1.2.
• Whereas the addition of small amounts of strong
base (OH-) combines with H+ provided by
equilibrium (Equation 1.1) moving to the left.
• In either case, change of H+ concentration, hence
pH, is unchanged.

4. From Equation 1.1, the dissociation constant (Ka) is defined as:
Ka =[H+][A-]/[HA]
pKa = -log Ka
= -log[H+][A-]/[HA]
= -log[H+]- log [A-]/[HA]
= pH-log [A-]/[HA]
pH = pKa+ log[basic form]/[acidic form]
This is the Henderson-Hassel balch equation.
The above equation is valid in the pH range of 3 to 11.
A buffer will have good buffering capacity when both forms are
present in a reasonable amount. So it is desirable to work within about
0.5 unit of the pKa.

6. Selection of an appropriate buffer
• Factors should be considered when choosing a
buffer:
• pKa and the effects of temperature
• interactions with other components (such as
enzymes and metal ions)
• compatibility with different purification
techniques
• ultraviolet (UV) absorption
• permeability through biological membranes
• cost

8. Examples
• For in gel permeation chromatography, almost any
buffer suitable for the protein of interest can be
chosen.
• anion-exchange chromatography, cationic buffers such
as Tris (and for cation-exchange chromatography,
anionic buffers such as phosphate) are preferred.
• However, these inorganic buffers do have some side
effects.
• Phosphate buffers are shown to inhibit many enzymes
including carboxypeptidase, urease, kinase, and
dehydrogenase.

9. Preparation of buffers
• In practice, the buffer is usually prepared at room
temperature.
• The pH of the working buffer should be tested after all
the components (e.g., ethylenediamine tetraacetic acid
[EDTA], dithiothreitol [DTT], Mg2+) have been added,
since the pH may change after such additions.
Unless otherwise stated, the pH of a buffer is adjusted
down with hydrochloric acid (HCl) and up with either
NaOH or KOH.

10. Cont…
• When the buffer requires the complete absence of metal
ions, the pH of the buffer should be adjusted with
tetramethyl ammonium hydroxide.
• Tris buffers should be avoided when a metal cofactor is
required for protein activity or stability; for 2 mM Mn2+
in100 mM Tris, 29% is found to be chelated by the buffer.
• Metal ion chelators such as EDTA are commonly used when
it is necessary to limit metal effects.
• One can make a buffer of the desired pH simply by mixing
components based on the available tables or calculations.
However, the pH of the final solution should be verified
with a pH meter.

11. 1.2 Use of Protease Inhibitors in Extraction
• Proteolysis can be a major problem after
extraction and at any stage of protein
purification.
The problems:
• complete inactivation of the desired protein,
generate degraded proteins, partially retaining
the biological activity.
• results in erroneous conclusions about the nature
of the protein (such as size and structure).

12. Cont…
Several classes of proteases are present in cells:
• Serine protease
• Cysteine (thiol) protease
• Aspartate (acidic) protease
• Metalloproteases protease

14. Mechanical Lysis for Protein Extraction
• Mechanical lysis: disruption of cells using
sonication, a pressure cell, homogenizer, or bead
beater.
• Mechanical lysis methods are economical and
preferable for large-scale preparations.
• However, mechanical lysis produces heat, which
needs to be controlled. Care should also be taken
to avoid foaming, to prevent surface denaturation
and oxidation.
• Mechanical lysis methods are two types: agitation
and liquid shear methods.

15. Homogenization
• Homogenization in a blender and in a Dounce are
common and simple procedures form disruption
soft tissues such as liver, heart, brain, and muscle.
• These methods are rapid (5 to 10 min) and gentle
to proteins.
• These homogenization procedures usually
produce heat, and thus the blender and the
associated container should be prechilled at 4℃.
• The homogenization should be performed a cold
room or on ice.

17. Sonication
• Sonication is most commonly used to disrupt
various types of cells (prokaryotes and
eukaryotes).
• Sonication creates vibrations that cause
mechanical shearing of the cell wall.
• Sonication is performed at the highest
allowable power setting, which is adjusted to
a level slightly below that at which foaming
occurs.

19. Disruption using French press
• In French press, cells are lysed at very high
pressure, followed by a sudden release to
atmospheric pressure.
• This rapid change in pressure causes cells to
burst.
• The appropriate volume of extraction in a French
press is about 10to 30 ml.
• The cell of the French press should be thoroughly
cleaned before and after use to prevent sample-
to-sample contamination.

21. Osmotic shock lysis
• Cell lysis may be achieved by osmotic shock
when suspended in a hypotonic solution (i.e.,
of a lower ionic strength than the cell
cytoplasm).
• Cells that are not protected by cell walls are
sensitive to osmotic stock. Red blood cells are
an example of this type.

22. Preparation of Extracts from
Prokaryotes
• Prokaryotes can be extracted by a variety of methods such
as enzymatic and mechanical lysis.
• Enzymatic lysis usually yields a lysate free of chromosomal
DNA.
• This is because lytic enzymes can create holes that are large
enough to escape proteins only.
• Extraction methods related to mechanical lysis cause a
release of nucleic acids, which should be removed from the
lysate because of viscosity problems and interference with
subsequent chromatographic steps.
• The most common method for removal of RNA and DNA is
the treatment of the lysate with protease-free RNAses and
DNAses.

23. Preparation of extracts from yeast
• The yeast cell wall can be digested with a variety of enzymes,
such as zymolyase, lyticase, and B-glucuronidase.
• The yeast cell wall contains the carbohydrate glucan,
mannoprotein, glycoprotein, and small amounts of chitin.
• Enzymatic reactions are usually carried out at room
temperature to 37℃ in the presence of sulfhydryl(thiol)
reagents in order to enhance the lysis.
• Lysis can be carried out directly, but in most instances
spheroplasts are prepared as an intermediate step with these
enzymes.
• Spheroplasts are then lysed in a variety of ways, such as
detergent extraction, homogenization using glass beads, or
French press

24. Preparation of Extracts from
Eukaryotes
• Several eukaryotes can be extracted by a variety
of pressure cells, such as the French press, Baton
press and Gaulin homogenizer.
• Extraction of eukaryotes by abrasive action of
glass beads remains a very effective method for
small to large scale.
• The percentage of cell breakage depends on the
speed of agitation.
• Sonication with glass beads is also effective.
• In all cases, beads are separated by
centrifugation or after decanting the supernatant.

25. Subcellular fractionation of animal
tissue
• Subcellullar fraction by centrifugation is a
widely used method for separating cellular
components.
• Subcellular fractionation is performed in three
steps:
• 1. tissue or cell suspension into a homogenate
• 2. separation of the components base on
density or sedimentation coefficient
• 3. analysis of the isolated fractions.

26. Cont..
• Tissue or cell suspension is accomplished by any
procedure previously described such as by
grinding, by sonication, and by using osmotic
properties.
• Suspension from soft tissues such as liver and
kidney is usually achieved with the homogenizer.
• Mincer and a blender such as a Waring blender is
used for tough tissues such as muscle

28. Cont…
• Tissue or cell disruption by osmotic methods is
applied mostly on red blood cells and
reticulocytes.
• Sonication is generally applied to disrupt bacterial
cells and some animal cells.
• Fractionation of these constituents can be
achieved by exploiting differences of their
physical and chemical properties.
• Centrifugation is the most widely used, probably
due to easy recovery of the sample at the end of
the fractionation procedure.

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