High Quality Lab Filtration Product at Axiva Sichem Biotech

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How to Choose Syringe Filter ?
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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.
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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)

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How to choose Membrane Filter ?
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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.

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TYPES OF BLOTTING TECHNIQUES
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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.

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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.

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Flow Diagram of Western Blot

• Protein Blot on
  SDS Polyacrylamide
• Nitrocellulose
  Gel Electrophoresis
• 
  
  

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• Label With Specific
  Detect Antibody
• Antibody
• 
  Reveals Protein
  
• 
  of Interest

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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.

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Definition

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

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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

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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.

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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.

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Most Uselful In

• This methods is used in the fields of
  molecular biology, Biochemistry, immunogenetics
  and other molecular biology disciplines.

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The Procedure Includes

• Tissue Preparation
• Gel Electrophoresis
• Transfer
• Blocking
• Detection
• Analysis

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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.

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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.

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Gel Electrophoresis Machine
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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.

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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.

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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.

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WESTERN BLOT REACTIVITY IN ONE HIV- 1,
SEROCONVERTER
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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.

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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.