Pseudomonas and Proteus: Partial Purification and Characterization of Antibacterial Protein

20 Pages·2017·0.3 MB·English

Int.J.Curr.Microbiol.App.Sci (2017) 6(12): 1349-1368
International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 6 Number 12 (2017) pp. 1349-1368

Journal homepage: http://www.ijcmas.com

Review Article https://doi.org/10.20546/ijcmas.2017.612.152

Pseudomonas and Proteus: Partial Purification and Characterization of
Antibacterial Protein: A Review

N.T. Eshwari* and M. Kannahi

Department of Microbiology, Sengamala Thayaar Educational Trust Women`s College,
Sundarakkotai, Mannargudi – 614001, Tamil Nadu, India
*Corresponding author

A B S T R A C T

Marine water samples were collected from a coastal region of Pushpavanam,

Nagapattinam District, Tamil Nadu, India. Marine Pseudomonas and Proteus were isolated

Key words and identified a ccording to morphology, biochemical test and selective complex marine
growth medium . The identified marine organisms were maintained with LB broth. The
Drop test assay, cultures were harvested by cooling centrifugation at 9,000x g for 20 min. This suspension
Prote in, was incubated at 28ºC for 24 hours after incubation the cells removed by centrifugation
Antib acterial method. The s upernatant was filter sterilized and antibacterial activity was zone of
activi ty, Colony inhibition performed by measuring overlay assay technique and drop test assay method.
formi ng unit, Gel Samples were suspended in sucrose – dye – solution and identification of antibacterial
electr ophoresis.
protein band resolved by polyacrylamide gel electrophoresis. The gel was washed with

A rticle Info distilled water. The protein fraction was identified by the drop test. All the proteins were
assayed with a bicinchoninic acid pro tein kit. The protein fraction elution was done with
A ccepted: phosphate buffer solution. The protei n fraction was stored at 20ºC after freeze – drying.
12 October 2017 The antibacterial protein was detect ed within SDS-PAGE treatment with Coomassie

Available Online: brilliant blue. The concentration level of antibacterial protein is necessary to result in cell
1 0 December 2017
death and to inhibit the growth of marine microorganisms in suitable medium. Bacterial

cells were inoculated at time zero and the bacterial growth rates and cell viability were

measured by colony forming unit.

Introduction

Marine organisms represent a promising The microbial diversity in the sea is yet to be
source for natural products of the future due revealed. In the last few years, marine
to the incredible diversity of chemical microorganisms emerged as a new field for
compounds that were isolated. Oceans cover the discovery of novel biologically active
almost 70% of the earth surface and over 90% compounds (Fenical, 1997).
of volume of its crust (Fenical, 1993). The
marine surface environment is a site of Marine natural products have an exceedingly
intense competition for living space by a wide bright future in the discovery of life saving
variety of organisms. Bacteria are generally drugs. The first antibiotic from marine
recognized as primary colonizers of this bacterium was identified and characterized
habitat (Bryers, 1982). (Burkholder et al., 1966).
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Int.J.Curr.Microbiol.App.Sci (2017) 6(12): 1349-1368
An increasing number of compounds with growing potential in biotechnological
antibacterial activity have recently been found applications (Mena and Gerba, 2009). Stanier
to be produced by a variety of organisms et al., 1966 published a comprehensive
present in the marine surface environment. appraisal of the taxonomy of Pseudomonas
The red alga to produce Delisea pulchra has spp., which is largely determined by
been shown to produce halogenated furanones phenotypes and biochemical capabilities
with activity against representatives of the (Mena and Gerba, 2009).
three major groups of fouling organisms,
including invertebrate larvae, macroalgae and The Pseudomonas was wide range of abiotic
bacteria (De Nys et al., 1995). and biotic environments which overlap among
the species. Pseudomonads are a large group
Bacteria are generally recognized as primary of free- living bacteria that live primarily in
colonizers of this habitat (Bryers and soil, seawater, and fresh water. Especially,
Characklis, 1982) and are able to rapidly form Pseudomonas aeruginosa is particularly
biofilms over freshly exposed surfaces as the prevalent in environments, such as soil, sea
prominence of bacteria during early water, sewage and associated with some
colonization events and their almost universal plants. Although commonly isolated from the
presence on marine surfaces, including those marine environment, the apparent distribution
of benthic marine invertebrates and algae has been restricted to river outfalls and
(Provasoli et al., 1980; Walls et al., 1993). shorelines (Velammal et al., 1994; Yoshpe –
Pure and Golderman, 1987; Kimata et al.,
Compared with terrestrial organisms, the 2004).
secondary metabolites produced by marine
organisms have more novel and unique The genus Proteus currently consists of five
structures owing to the complex living species: P. mirabilis, P. vulgaris, P. penneri,
circumstance and diversity of species, and the P. hauseri and P. myxofaciens, as well as
bioactivities are much stronger (Burgess et three unnamed Proteus genomospecies
al., 1999; Proksch et al., 2002; Schupp et al., (Ohara et al., 2000). Proteus myxofaciens is
1999). the only Proteus species without any
significance in the pathogenicity of humans, it
Furthermore, along with the deep studies of has been isolated from living and dead larvae
marine natural products biosynthesis, some of the gypsy moth Proteria dispar (Janda et
evidence indicates that many bioactive al., 2006).
compounds previously found in marine
animals and plants were produced or Marine organisms
metabolized by associated microorganisms
(Carte et al., 1996; Kohler et al., 1999; Screening of organic extracts from marine
Osinga et al., 2001; Rinehart, 2000; Sponga et sponges and other marine organisms is a
al., 1999). common approach to identify compounds of
biomedical importance. Notable exception to
Members of the genus Pseudomonas show this is the study performed by Mebs et al.,
remarkable metabolic and physiological (1985) and Kristina et al., (1997). Aqueous
versatility, enabling their colonization of extracts from sponge species were tested for
diverse terrestrial and aquatic habitats. hemolytic hemagglutinating, cytotoxic,
Pseudomonas are of great interest because of antimicrobial, anticholesterase and lethal
their role in plant and human disease and their activities.
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The genus Pseudoalteromonas includes both agriculture. Most of them are produced by
pigmented and non-pigmented, Gram- Streptomycetes (Demain, 1999). Most
negative, rod shaped, heterotrophic marine streptomycetes and also other actinobacteria
bacteria that are motile by means of polar produce a diverse array of antibiotics,
flagella. This recently described genus is the including aminoglycosides, macrolides,
result of extensive taxonomic revision on the peptides, polyenes, polyether, tetracyclines,
basis of the phylogenetic relationships among etc. In searching for new antibiotics, over
members of the genera Alteromonas, 1000 different bacteria (including
Shewanella and Moritella. Gauthier et al., actinobacteria), fungi and algae have been
(1995) suggested that the genus Alteromonas investigated. To prevent exponential
be divided into two genera, with the majority emergence of microorganisms becoming
of species being reclassified within the new resistant to the clinically available antibiotics
genus Pseudoalteromonas, leaving already marketed, the periodic replacement of
Alteromonas macleodii as the sole species of existing antibiotics is necessary.
Alteromonas. Species of the genus
Pseudoalteromonas, the majority of which Marine fungi are an ecological rather than a
appear to be associated with eukaryotic hosts taxonomic group. However, the ascomycetes
(Holmstrom and Kjelleberg, 1999), are order Halosphaeriales comprises largely
isolated frequently from marine waters around marine species with 43 genera and some 133
the world. Species have been isolated from species (Jones, 1995). This is approximately
various animals such as tunicates (Holmstrom half of the total number of marine
et al., 1998), mussels (Ivanova et al., 1996), ascomycetes (Kohlmeyer and Volkmann –
pufferfish (Simidu et al., 1990) and sponges Kohlmeyer, 1991).
(Ivanova et al., 1998) and from a range of
marine algae (Akagawa - Matsushita et al., Kohlmeyer and Kohlmeyer (1979) stated that
1992; Yoshikawa et al., 1997). “considerable additions of new species in the
future are unlikely” and indicated that there
The bacterial strains in this study were were then less than 500 marine fungi. Several
isolated from the surface of the marine alga recent papers have addressed the issue of the
Ulva lactuca. Both strains have been shown diversity and numbers of marine fungi (Jones,
to inhibit the settlement of larvae of the 1995; Jones and Mitchell, 1996; Jones and
marine invertebrate Balanus amphitrite and Alias, 1997). Jones and Mitchell (1996)
the germination of spores of the green alga U. estimated that there are 1500 species
lactuca and a species of the red alga compared with 200,000 marine animals and
Polysiphonia (Egan et al., 2000). 20,000 marine plants. The dramatic rise in the
numbers of new marine fungi, which has
Streptomycetes, Gram negative filamentous occurred since the statement by Kohlmeyer
bacteria, are widely distributed in a variety of and Kohlmeyer (1979), is due to the nature of
natural and man-made environments and the substrata examined for the occurrence of
constitute a significant component of the fungi. Kohlmeyer and Kohlmeyer (1979)
microbial population in most soil (Watve et listed 42 species of mangrove fungi, while the
al., 2001). The results of extensive screening current number approaches 200 species (Jones
have been the discovery of about 4000 and Alias, 1997). Similarly the number of
antibiotic substances from bacteria and fungi, fungi recorded by Kohlmeyer and coworkers
many of which have found applications in from Junces roemerianus has also increased
human medicine, veterinary medicine and (Kohlmeyer et al., 1996).
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Actinomycetes are the most widely distributed activities (Patterson et al., 1994; Reichenbach
groups of microorganisms in nature. They are et al., 2001; Schwart et al., 1990; Shimkets et
attractive, bodacious and charming al., 2004). Recently, the rate of discovery of
filamentous gram-positive bacteria. They new compounds from existing genera
make up in many cases, especially under dry obtained from terrestrial sources has
alkaline conditions, a large part of the decreased, while the rate of reisolation of
microbial population of the soil (Athalye et known compounds has increased. Moreover,
al., 1981; Goodfellow and Williams, 1983; the rise in the number of drug-resistant
Lacey, 1973; Lacey, 1997; Nakayama, 1981; pathogens and the limited success of
Waksman, 1961). Based on several studies strategies such as combinatorial chemistry in
among bacteria, the actinomycetes are providing new agents indicate an uncertain
noteworthy as antibiotic producers, making forecast for future antimicrobial therapy
three quarters of all known products; the (Projan, 2003; Projan et al., 2002). Thus, it is
Streptomyces are especially prolific (Lacey, critical that new groups of microbes from
1973; Lechevalier, 1989; Locci, 1989; unexplored habitats be pursued as sources of
Saadoun and Gharaibeh, 2003; Waksman, novel antibiotics and other small-molecule
1961). therapeutic agents (Bull et al., 2000).

Actinomycetes are the most economically and Physico – chemical parameters of marine
biotechnologically valuable prokaryotes and environment
are responsible for the production of about
half of the discovered secondary metabolites. Analysis of the physico - chemical parameters
Because of the excellent track record of of the marine water samples collected from
actinomycetes in regard a significant amount different sites. The parameters such as PH,
of effort has been focused on the successful temperature, electrical conductivity, dissolved
isolation of novel actinomycetes from solids, salinity, zinc, copper, iron, nickel,
terrestrial sources for drug screening cobalt, total mercury, total cyanide, total lead,
programmes in the past fifty years. Recently selenium, total silver, nitrate, nitrite,
the rate of discovery of new compounds from ammonia, inorganic sulphide and sulphate
terrestrial actinomycetes has decreased were analyzed using the standard methods
whereas the rate of reisolation of known (Strickland and Parsons, 1972).
compounds has increased. Thus, it is excited
that new groups of actinomycetes from The collected sediment sample were first air
unexplored or under exploited habitals be dried at room temperature, then crushed using
persued as sources of novel bioactive a porcelain mortar and pestle and then sieved
secondary metabolites (Donia et al., 2003). for further analysis. The PH of the suspension
was read using PH meter (Systronics, India),
The diversity of actinomycete secondary to find out the soil PH. Electrical conductivity
metabolites is unrivaled and unmatched in of the soil was determined in the filtrate of the
medical significance. Structurally and water extract using Conductivity Bridge and
functionally diverse bioactive compounds Cation Exchange Capacity (CEC) of the soil
have also been isolated from other was determined by using 1 N ammonium
prokaryotes, including members of the acetate solution. The reagents used for the
myxobacteria (e.g., Sorangium) and analysis were AR grade and double distilled
cyanobacteria (e.g., Nostoc), as antibiotics water was used for preparation of solutions.
with antimicrobial, antiviral, and antitumor The analyzed Physico - chemical parameters
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were observed by standard methods TA = total number of samples.
(Muthuvel and Udayasoorian, 1999).
Values were considered according to the
Bacterial enumeration following classification:

Bacteria were enumerated by the acridine <10% = Rare, 10 < 25 % = Low frequency 25
orange direct count (AODC) method (Hobbie < 35% = Frequent, 35< 50% = Abundant and
et al., 1977). Bacteria were sized from > 50% Very abundant.
enlarged photomicrographs (~ 4300) using a
digitizer (Hewlett Packard 9874A) according Enumeration of actinomycetes
to Simon (1987). Usually 40 to 120 cells were
sized on each filter. Isolation and enumeration of actinomycetes
were performed by soil dilution plate
To compute the cell volume we considered technique using Glycerol-Yeast Extract Agar
the rod-shaped cells to be cylinders with a (Glycerol 1 g, Yeast extract 0.4 g, K2HPO4
hemispherical cap and cocci to be spheres. No 0.02 g, Peptone 5.0 g, Agar 3 g, Distilled
correction was made for possible cell water 200 ml) complemented with nystatin
shrinkage as a result of sample preparation. (50 µg/ml) at 27ºC (Waksman, 1961). One
gram of dried soil was taken in 9 ml of
The bacterial growth rate was calculated by distilled water, agitated vigorously and
the equation for exponential growth: preheated at 500 C for 0.5 hours. Different
aqueous dilutions, 10-3, 10-5, and 10-7 of the
µ = (lnN2 – lnN1) / (t1 - t2) suspension were applied onto plates and 20
ml of melted medium at around 50ºC was
Where N and N = bacterial abundance at the
1 2 added to it. After gently rotation, the plates
beginning and the end of an incubation
were incubated at 27oC for 7 to 14 days.
interval; t and t = the corresponding times.
1 2 Selected colonies (rough, chalky) of

actinomycetes were transferred from mixed
Fungal Enumeration (Dajoz, 1983)
culture of the plates onto respective agar

plates and incubated at 27ºC for 7 days. Plates
Total Spore Count (TVC) of aquatic samples
containing pure cultures were stored at 40ºC
was calculated by following pour plate
until further examinations.
technique. Each sample was serially dilluted

with dilutions ranging from 10-1 - 10-9 and 1ml
Isolation and identification of marine
of each dilution were poured on the nutrient
organisms
agar plates. Plates were incubated at 37' c for

72 hours in an inverted position and the
One gram from each soil sample was placed
colonies were counted. The count was
in 9 ml of asparagine broth enrichment
expressed as the number of colony forming
medium consisting of 2g l-1 asparagine L-
units in 1 ml of the original sample. The
monohydrate (Fluka, Switzerland) 1g l-1
occurrence of fungal species was calculated
K HPO (BDH, England) and 0.5 g l-1
2 4
according to Dajoz (1983), using the formula;
MgSO ⋅7H O (BDH, UK) in order to
4 2
Fo = Ta. 100/ TA enhance Pseudomonas growth. The samples
were incubated for 48 h at 37ºC with vigorous
Where,
shaking at 200 rpm to provide aeration for the
Ta = number of samples in which a taxon has
bacteria. A loopful of the resulting bacterial
occurred.
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suspension was streaked onto asparagine immersion (100x) (Han’s Christian Gram,
plates containing 1.5% agar (Oxoid, UK) and 1884).
incubated at 37ºC until colonies developed.
The bacteria were then transferred to fresh Motility test
asparagine plates according to the
morphological characteristics of colony The motility was situated by employing
colour, shape and size (Projan, 2002). hanging drop method. A drop of culture broth
was placed on the centre of the cover slip.
Actinomycetes were isolated by soil dilution The vaseline was applied over each corner
plate technique on starch casein agar medium, slip. Then the slide was observed through the
starch nitrate agar medium, glycerol glycine oil immersion microscope (Jordan et al.,
agar medium, and chitin agar medium 1934).
(Haefner et al., 2003). The plates were
incubated at 28°C and the number of colonies Biochemical characterization
was determined after 15 days. All the medium
containing 50% sea water was supplemented A single colony was selected for biochemical
with Nystatin 50μg/ml and Nalidixidic acid tests. P. aeruginosa is oxidase positive,
20 μg/ml to inhibit the bacterial and fungal hydrolyses casein, and produces pyocyanin
contamination, respectively (Haque et al., and/or fluorescence. Occasionally a non-
1992). pigmented variant of P. aeruginosa can occur,
therefore a pyocyanin negative, casein
The selected colonies were picked up and hydrolysis positive, fluorescence positive
further purified by streak plate technique over culture can also be regarded as P. aeruginosa.
starch casein agar slants. The antimicrobial Isolated strains were biochemically identified
activity was studied preliminarily by cup plate by conventional tests followed by use of API
method (Haque et al., 1992) against bacteria 20 NE identification system (API 20 NE,
and fungi. The test organisms were used are Biomerieux).
Bacillus subtilis, Staphylococcus aureus
Escherichia coli, Pseudomonas aeruginosa, Colony characteristics, including pigment
Aspergillus niger. After preliminary testing of production were determined on Pseudomonas
the isolates for their antimicrobial activities, P Agar (Acumedia) and NA (Difco). All
the most active isolates were selected for strains were characterized by the following
further study. classical tests according to Bergey’s Manual
of Systematic Bacteriology (Palleroni, 1984)
Gram staining Gram staining, cytochrome oxidase
production, catalase production and growth
A smear of the isolate was prepared on a on McConkey Agar and 42°C. Isolated strains
clean glass slide and smear was allowed to air were biochemically identified by
dry and then heat fixed. Heat fixed smear was conventional tests followed by use of API 20
flooded with crystal violet and after one NE identification system (API 20 NE,
minute, it was washed with water and flooded Biomerieux). To examine the enzyme profiles
with mordant Gram’s iodine. Smear was of isolates the API ZYM (Biomerieux) system
decolorized with 95% ethyl alcohol, and then was used, essentially as recommended by the
washed with water. The smear was counter supplier. The results were obtained in
stained with safranin for 45 seconds. After duplicate and analyzed employing the Apilab
washing with water, smear was dried with Plus Software (Biomerieux).
tissue paper and examined under oil
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Morphological characterization sodium dodecyl sulfate (SDS) to separate
them exclusively according to molecular
Morphological and cultural characters of the weight. This technique was firstly introduced
selected actinomycetes strain was studied by by Shapiro et al., (1967). SDS is a reducing
inoculating into sterile International agent that breaks disulfide bonds, separating
Streptomycetes Project ISP 1,3-7,9 media the protein into its sub-units and also gives a
(Matson et al., 1989; Ohshima et al., 1991). net negative charge which allows them to
The media were sterilized and poured into migrate through the gel in direct relation to
sterile petridishes. After solidification of the their size. In addition, denaturation makes
media, culture of the selected strain was them lose their tertiary structure and therefore
streaked on the media surface by simple migration velocity is proportional to the size
method aseptically and incubated at 37o C for and not to tertiary structure.
7 days (Salle et al., 1948; Shomura et al.,
1987). Morphological characters such as Some highlights of the polyacrylamide gel
colony characteristics, type of aerial hyphae, electrophoresis are:
growth of vegetative hyphae, fragmentation
pattern and spore formation were observed. Gels suppress the thermal convection caused
by application of the electric field, and can
Molecular characterization also act as a sieving medium, retarding the
passage of molecules; gels can also simply
SDS – PAGE (Sodium Dodecyl Sulfate – serve to maintain the finished separation, so
Poly Acrylamide Gel Electrophoresis) that a post electrophoresis stain can be applied

Gel electrophoresis of proteins with a The polyacrylamide gels are formed by
polyacrylamide matrix, commonly called polymerization of acrylamide by the action of
polyacrylamide gel electrophoresis (PAGE) is a cross-linking agent, the bis-acrylamide, in
undoubtedly one of the most widely used the presence of an initiator and a catalyst.
techniques to characterize complex protein Persulfate ion (S2O8), that is added as
mixtures. It is a convenient, fast and ammonium persulfate (APS) is the
expensive method because they require only gelsolidifying initiator and a source of free
the order of micrograms quantities of protein. radicals, while TEMED (N, N, N', N'-
The proteins have a net electrical charge if tetramethylethylenediamine) catalyzes the
they are in a medium having a pH different polymerization reaction by stabilizingthese
from their isoelectric point and therefore have free radicals. In some situations, for example,
the ability to move when subjected to an isoelectric focusing the presence of persulfate
electric field. The migration velocity is can interfere with electrophoresis, so
proportional to the ratio between the charges ribofavin and TEMED are used instead
of the protein and its mass. The higher charge
per unit of mass the faster the migration. Acrylamide solutions are degassed as oxygen
Proteins do not have a predictable structure as is an inhibitor of polymerization. Moreover,
nucleic acids, and thus their rates of migration the polymerization releases heat that could
are not similar to each other. They can even cause the formation of bubbles within the gel
not migrate when applying an electromotive
force (when they are in their isoelectric The rate of polymerization is determined by
point). In these cases, the proteins are the concentration of persulfate (catalyst) and
denatured by adding a detergent such as TEMED (initiator)
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The acrylamide gel electrophoresis systems merkapto etanol, 5ml; glycerol, 10ml;
may be performed using one or more buffers, bromophenol blue, 1mg) and added 20%
in these cases of continuous phosphate buffer SDS, 0.1ml, heated for 10 min at 95˚C.
system (Weber and Osborn, 1968) or Protein extracts underwent SDS-PAGE on
discontinuous buffer systems (Ornstein, 1964; vertical slab gels in an electrophoresis
Davis, 1964: Laemmli, 1970) adopted the apparatus. Electrophoresis was performed
discontinuous electrophoresis method and the using a power supply operated at a constant
term "Laemmli buffer" is often used to current of 20 mA through the stacking gel and
describe the tris-glycine buffer system that is 30 mA through the resolving gel. A standard
utilized during SDS-PAGE. In discontinuous protein solution (MBI Fermentas, SM 0439),
systems the first buffer ensures the migration containing 7 proteins ranging in size from
of all proteins in the front of migration, what 14.4 to 116 kDa, was used as a molecular
causes the accumulation of the entire sample weight marker and for the normalization and
that has been loaded into the well. The interpolation of the protein patterns. The
separation really begins from the moment proteins were run on 10% SDS-PAGE and
when the migration front reaches the gels were stained with Coomassie blue R250.
boundary of the second buffer. The first gel, Afterwards, the gels were distained until
“stacking”, has larger pore (lower percentage protein bands become clearly visible.
of acrylamide / bisacrylamide) and has a pH
more acidic than the second gel which is what Protein separation
really separates proteins. This system is
particularly suitable for analyzing samples Proteins separated on a polyacrylamide gel
diluted without losing resolution can be detected by various methods, for
(Westermeier, 2005). instance dyes and silver staining.

Sodium dodecyl sulfate–polyacrylamide gel Dyes
electrophoresis (SDS–PAGE) The extraction
of total cell protein content of the strains The Coomassie blue staining allows detecting
studied was determined according to Pot et up to 0.2 to 0.6 μg of protein, and is
al., (1994) and separation of proteins based on quantitative (linear) up to 15 to 20 μg. It is
their molecular weight was achieved through often used in methanol-acetic acid solutions
SDS-PAGE technique (Laemmli, 1970). and is discolored in isopropanol-acetic acid
solutions. For staining of 2-DE gels it is
The cultures were used as an inoculum (1% recommended to remove ampholytes by
v/v) in 10 ml NB, and incubated at 37 ˚C for adding trichloroacetic (TCA) to the dye and
24 h. subsequently discolor with acetic acid.

Silver staining
The pellet washed two or three times with
sodium phosphate buffer (0.01M) containing It is an alternative to routine staining protein
0.8 % NaCl (pH 7.3; sodium phosphate- gels (as well as nucleic acids and
buffered saline). The extracts were sonicated lipopolysaccharides) because its ease use and
on ice during 45 seconds. After lysis, the high sensitivity (50 to 100 times more
sonicated extracts were centrifuged at 5000xg sensitive than Coomassie blue staining). This
for 10 min. The pellet was discarded and the staining technique is particularly suitable for
supernatant was supplemented with sample two-dimensional gels.
buffer (0.062 M Tris- HCl, 0.75 g; 2-
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Int.J.Curr.Microbiol.App.Sci (2017) 6(12): 1349-1368
Detection of radioactive proteins by the membrane. The absorption of the drop
autoradiography causes the adhesion of the protein to the
membrane, leaving it as a spot or “dot” (this
The autoradiography is a detection technique is the case of the “dot blot”). There are
of radioactively labeled molecules that uses devices that make possible the application of
photographic emulsions sensitive to proteins to membrane directly, using a suction
radioactive particles or light produced by an that facilitates the penetration of the solution,
intermediate molecule. and are named “dot blot” or “slot blot” on the
basis that the proteins were applied as a
The emulsion containing silver is sensitive to circular drop or a line.
particulate radiation (alpha, beta) or
electromagnetic radiation (gamma, light...), so Working with proteins bound to a membrane
that it precipitates as metallic silver. The has advantages over employment within the
emulsion will develop as dark precipitates in gel:
the region in which radioactive proteins are
detected. Staining and discolor are faster

Western blot No staining occurs to ampholytes in
isoelectric focusing gels
Western blot is a widely used method in
molecular biology and biochemistry to detect Smaller amounts of proteins are detected as
proteins in a sample of cell homogenate or they are concentrated at the surface and not
extract. The proteins are transferred from the diluted across the thickness of the gel
gel to a membrane -made of nitrocellulose,
nylon or polyvinylidene difluoride (PVDF), The membranes are much easier to
where they are examined using specific manipulate than the gel itself
antibodies to the protein. As a result, the
amount of protein in a sample can be Blotting procedure
examined and it is also possible to compare
levels among various analytical groups. It consists of 5 stages:

The method was initiated in the laboratory of Immobilization of proteins on the membrane
George Stark at Stanford. The name "western was either by transference (electrophoresis,
blot" was given to the technique by Burnette suction, pressure...) or by direct application.
(1981), comparing it with the "southern blot" The procedure starts piling a flat sponge on
technique for DNA detection developed by filter paper soaked in transference buffer, the
Edwin Southern. The detection of RNA is gel, the membrane in direct contact with the
also called Northern blotting. gel plus filter paper and finally a flat sponge.
This set is included between two layers of
The most powerful method is the transference perforated plastic and placed in a tank which
of proteins from the gel to a membrane by is a saline solution (transference buffer) and
applying an electric field perpendicular to the two plate electrodes (designed to achieve a
gel. There are however other methods of uniform field across the surface of the gel).
transferring or applying a protein on the They are disposed so that the gel is toward the
membrane. The simplest is to apply it directly anode (-) and the membrane to the cathode
as a small drop of a concentrated solution on (+).
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Int.J.Curr.Microbiol.App.Sci (2017) 6(12): 1349-1368
Saturation of all binding sites of proteins in first, database search program. While very
the membrane not occupied to prevent successful in terms of sensitivity, it is quite
nonspecific binding of antibodies, which are slow to process data and there are concerns
proteins. against specificity, especially if multiple
posttranslational modifications (PTMs) are
Incubation with primary antibody against the present (Jimmy et al., 1994).
protein of interest.
Mascot - is a powerful search engine that uses
Incubation with secondary antibodies, or mass spectrometry data to identify proteins
reactives acting as ligands of the primary from primary sequence databases
antibody bound to enzymes or other markers. (www.matrixscience.com) (Perkins et al.,
Enzyme coupled secondary antibodies: an 1999).
antibody to the specific binding antibody is
conjugated to the enzyme peroxidase or Scaffold 3 – is a software which produces a
alkaline phosphatase (Blake et al., 1984). confidence level for protein identification
from one or more Mascot, Sequest, X!
Another possibility is the use of an amplifying Tandem, or Phenyx searches. It can be used in
enzyme which is part of a biotin-avidin- conjunction with a MS/MS search engine in
peroxidase complex (Hsu et al., 1981) or a order to validate/visualize data across
complex with alkaline phospohatase. multiple experimental runs as well as provide
a more accurate protein probability
Enhanced chemiluminescence (ECL) is other
commonly used method for protein detection Purification of protein from Pseudomonas
in western blots (Laing, 1986). ECL is based and Proteus
on the emission of light during the horse
radish peroxides (HRP) - and hydrogen The protein concentrations of the samples
peroxide-catalyzed oxidation of luminol. The recovered in the extraction and purification
emitted light is captured on film or by a CCD procedures were estimated by the method of
camera, for qualitative or semi- quantitative Lowry (Lowry et al., 1951), with bovine
analysis. This method allows stripping and re- serum albumin as standard, and total
probing the blot with different antibodies. carbohydrates were determined by the phenol
– sulfuric acid procedure of Dubois (Dubois
Software and database search algorithms et al., 1956), with glucose as standard.
to analyze spectral data
The antibacterial substance from the active
A variety of tools and commercially available bacteria supernatant was purified by
software exist that allow for protein ammonium sulfate precipitation and High –
identification from peptide sequences Performance Liquid Chromatography
determined by mass spectrometry. (HPLC). First the purified substance was
obtained, and then solid ammonium sulfate
Some examples of database search programs was added slowly to 85% and 50% saturation
and algorithms are: at 4 ºC with constant stirring (10,000 x g for
30 min at 4ºC) and resuspended in H O.
2
SEQUEST - identifies collections of tandem These samples were then loaded onto Agilent
mass spectra to peptide sequences that have Zorbax 300 SB – C18 column for HPLC.
been generated from databases of protein Elution was performed with 95%, 50%, 5%
sequences. It was one of the first, if not the
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