High Quality Lab Filtration Product at Axiva Sichem Biotech - PowerPoint PPT Presentation

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High Quality Lab Filtration Product at Axiva Sichem Biotech

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Axiva Sichem Biotech is offering high quality Lab Filtration Products (syringe filters, membrane filters, glass fiber filters, filter papers) that are manufactured and developed by our highly experienced team as per market trends. Further, our quality controllers check these products on the basis of their basic weight, thickness, air flow and mechanical strength. We offer these products in different specifications to our valuable customers, spread across the country. – PowerPoint PPT presentation

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Title: High Quality Lab Filtration Product at Axiva Sichem Biotech


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
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
Tissue Preparations
  • 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.
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