1.
How to Choose Syringe Filter ?

2.
SYRINGE FILTERS
A syringe filters generally consists of a plastic housing with a membrane which
serves as a filter. The fluid to be purified may be cleaned by drawing it up the
syringe through the filter, or by focusing the unfiltered fluid out through the filter.
Membrane Filters
Membrane Filters or membrane is microporus films with specific filtration rating.
Membrane retains particles and micro organisms that exceed their filtration rating
by acting as a physical barrier and capturing such particles on the surface of
membrane.

3.
Case study
 Chloroform (Halogenated Hydro carbon)
 DMSO (Dimethyl Sulfoxide)
 Lipid solution
 Hormones
 IVF / NMR
 Non – aqueous colored solutions
 Highly viscous aqueous solution (Plasdone XL,
kolldon SR, Xanthan gum, Avicel PH 112)

4.
How to choose Membrane Filter ?

5.
What is blotting ?
Blotting are techniques for transferring DNA, RNA
and proteins onto a carrier so they can be separated,
and often follows the use of a gel electrophoresis. The
southern blot is used for transferring DNA, the
Northern blot for RNA and the western blot for
PROTEIN.

6.
TYPES OF BLOTTING TECHNIQUES
Blotting Technique
Southern Blot
It is used to
detect DNA.
Northern Blot
It is used to
detect RNA.
Western Blot
It is used to
detect protein.

7.
Protein Blotting
Protein blotting is an
analytical Method that
involves the immobilization
Of proteins on membranes
before detection Using
monoclonal or polyclonal
antibodies.
There are different blotting
protocols (dot blot, 2D blot);
one of the most powerful is
western blotting.

8.
Principle of Western Blotting
 Western blotting is an Immunoblotting
technique which rely on the specificity of binding
between a molecule of interest and a probe to allow
detection of the molecule of interest in a mixture of
many other similar molecules.
 In Western blotting, the molecule of interest is a
protein and the probe is typically an antibody raised
against that particular protein.
 The SDS PAGE technique is a prerequisite for
Western blotting.

9.
Flow Diagram of Western Blot
Protein Blot on SDS Polyacrylamide
Nitrocellulose Gel Electrophoresis

10.
Label With Specific Detect Antibody
Antibody
Reveals Protein
of Interest

11.
Western Blot
(Immunoblotting)
A technique for
detecting specific
proteins separated by
electrophoresis by use
of labeled antibodies.
So called Since it has
some similarity to a
southern blot.

12.
Definition
The Western Blot is
an analytical
technique used to
detect specific
proteins in a given
sample of tissue
homogenate or
extract.

13.
Advantages of Western
Blot
Western blot analysis
can analyze any protein
sample whether from
cells or tissues, but
also can analyze
recombinant proteins
synthesized in vitro.
Western blot is
dependent on the
quality of antibody you
use to probe for your
protein of interest, and
how specific it is for
this protein

14.
Gel Electrophoresis
Uses gel electrophoresis to
separate native or denatured
proteins by the length of the
polypeptide (denaturing
conditions) or by the 3-D
structure of the protein (native /
non-denaturing conditions). The
proteins are then transferred to a
membrane (typically
nitrocellulose or PVDF), where
they are probed (detected) using
antibodies specific to the target
protein.

15.
Monoclonal and Polyclonal
Antibodies are used
Here are now many
reagent companies that
specialize in providing
antibodies (both
monoclonal and
polyclonal antibodies )
Against tens of
thousands of different
proteins.

16.
Most Uselful In…
This methods is
used in the fields
of molecular
biology,
Biochemistry,
immunogenetics
and other
molecular
biology
disciplines.

17.
The Procedure Includes
Tissue Preparation
Gel
Electrophoresis
Transfer
Blocking
Detection
Analysis

18.
Samples may be taken from whole tissue or from cell culture.
In most cases, solid tissues are first broken down mechanically using a
blender.
It should be noted that bacteria, virus or environmental samples can be the
source of protein and thus Western blotting is not restricted to cellular studies
only.
Assorted detergents, Salts, and buffers may be employed to encourage lysis of
cells and to solubilize proteins.
Tissue preparation is often done at cold temperature to avoid protein
denaturing.

19.
Gel Electrophoresis
The proteins of the samples are separated using gel
electrophoresis. Separation of proteins may be by isoelectric
point molecular weight, electric charge, or a combination of
these factors.
The Principle involved is the difference in the
ELECTROPHORETIC
MOBILITIES of different proteins.

20.
Gel Electrophoresis Machine

21.
Transferring
In Order to make the proteins accessible to antibody
detection, they are moved from within the gel onto a membrane made of nitrocellulose
or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a
stack of filter papers placed on top of that. The entire stack is placed in a buffer solution
which moves up the paper by capillary action, bringing the proteins with it.
Another method for transferring the proteins is called electro blotting and uses an
electric current to pull proteins from the gel onto the PVDF or nitrocellulose
membrane.

22.
Blocking
The membrane has the ability to bind to proteins in this case both the target and
antibodies are proteins and so there could be some unwanted binding.
Blocking of non-specific binding is achieved by placing the membrane in a dilute
solution of protein – typically Bovine serum albumin (BSA) with a minute percentage
of detergent such as tween 20.
The protein in the dilute solution attaches to the membrane in all places where the
target proteins have not attached. Thus, when the antibody is added, there is no room on
the membrane for it to attach other than on the binding sites of the specific target
protein.

23.
Detection
During the detection process, the
membrane is “Probed” for the
protein of interest with a modified
antibody which is linked to a
reporter enzyme, which when
exposed to an appropriate substrate
drives a colorimetric reaction and
produces a color.

24.
WESTERN BLOT REACTIVITY IN ONE HIV- 1, SEROCONVERTER

25.
Analysis
After the unbound probes are washed away, the western blot is ready
for detection of the probes, that are labeled and bound to the protein of
interest.
Size approximations are taken by comparing the stained bands to that
of the marker loaded during electrophoresis.
The process is repeated for a structural protein, such as actin, or tubulin
that should not change between samples.

26.
Advantages
While ELISA being a non-specific test, Western blotting is a
more specific test for detection of HIV.
It can detect one protein in a mixture of proteins while giving
information about the size of the protein and so is more specific.
Western blot test is referred to as the ‘Gold standard’
It also tells you how much protein has accumulated in cells.

27.
Western Blot in Clinical Medicine
The confirmatory HIV test employs a Western blot to detect anti-HIV antibody in a
human serum sample. Proteins from known HIV-infected cells are separated and
blotted on a membrane then, the serum to be tested is applied in the primary antibody
incubation step; free antibody is washed away, and a secondary anti-human antibody
linked to an enzyme signal is added . The stained bands then indicate the proteins to
which the patient’s serum contains antibody.
A western blot is also used as the definitive test for Bovine Spongiform encephalopathy
(BSE, commonly referred to as ‘mad cow disease’)
Some forms of Lyme disease testing employ Western Blotting.

28.
Western Blot a Confirmatory test
in HIV Infection
The virus is enveloped
with different
The detection of these
proteins are useful in the
detection of the presence
of the virus.
Western Blotting helps in
the detection of these
proteins.

29.
Contact Us
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