Western blotting

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  • 1. BY AKHIL CHINNABATHINI 11IMMM03WESTERN BLOTTING
  • 2. CONTENTS Tissue preparation Gel electrophoresis Transfer Blocking Detection Analysis Applications
  • 3. INTRODUCTION The western blot (sometimes called the protein immunoblot) is a widely accepted analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide. The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are stained with antibodies specific to the target protein.
  • 4. TISSUE PREPARATION Samples can be taken from whole tissue or from cell culture. Solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer(smaller volumes), or by sonication. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins
  • 5. GEL ELECTROPHORESIS The proteins of the sample are separated using gel electrophoresis We use SDS-PAGE(sodiumdodecyl sulphate-polyacrylamide gelelectrophoresis).
  • 6. *Sampledproteins becomecovered in thenegativelycharged SDSand move to thepositivelychargedelectrodethroughthe acrylamidemesh of the gel.
  • 7. SDS-PAGE (POLYACRYLAMIDE GELELECTROPHORESIS) SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique widely used in biochemistry, forensics, genetics and molecular biology: to separate proteins according to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight). to separate proteins according to their size, and no other physical feature.
  • 8. ..SDS SDS (the detergent soap) breaks up hydrophobic areas and coats proteins with negative charges thus overwhelming positive charges in the protein. The detergent binds to hydrophobic regions in a constant ratio of about 1.4 g of SDS per gram of protein. Therefore, if a cell is incubated with SDS, the membranes will be dissolved, all the proteins will be solubalized by the detergent and all the proteins will be covered with many negative charges.
  • 9. PAGE If the proteins are denatured and put into an electric field (only), they will all move towards the positive pole at the same rate, with no separation by size. However, if the proteins are put into an environment that will allow different sized proteins to move at different rates. The environment is polyacrylamide. the entire process is called polyacrylamide gel electrophoresis
  • 10. ..PAGE Small molecules move through the polyacrylamide forest faster than big molecules. Big molecules stays near the well.
  • 11. TRANSFER The proteins are transferred to a sheet of special blotting paper called nitrocellulose. The proteins retain thesame pattern of separation they had on the gel. In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polvinylidene difluoride (PVDF).
  • 12. …TRANSFER The primary method for transferring the proteins is called electro blotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dyes
  • 13. Protein gel (SDS-PAGE) that has been stainedwith Coomassie Blue.
  • 14. BLOCKING Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein - typically 3-5% Bovine serum albumin (BSA) or non-fat dry milk (both are inexpensive) in Tris- Buffered Saline (TBS), with a minute percentage of detergent such as Tween 20 or Triton X-100.
  • 15.  when the antibodyis added, there is noroom on the membranefor it to attach otherthan on the bindingsites of the specifictarget protein.
  • 16. DETECTIONThere are two steps for the detection of the proteinPrimary antibody After blocking, a dilute solution of primary antibody (generally between 0.5 and 5 micrograms/mL) is incubated with the membrane under gentle agitation The solution is composed of buffered saline solution with a small percentage of detergent, and sometimes with powdered milk or BSA. The antibody solution and the membrane can be sealed and incubated together for anywhere from 30 minutes to overnight.
  • 17. Secondary antibody After rinsing the membrane to remove unbound primary antibody, the membrane is exposed to another antibody, directed at a species-specific portion of the primary antibody. Several secondary antibodies will bind to one primary antibody and enhance the signal.
  • 18. ANALYSIScalorimetric detectionThe colorimetric detection method depends on incubation of the western blot with a substrate that reacts with the reporter enzyme (such as peroxidase)Chemiluminescent detectionChemiluminescent detection methods depend on incubation of the western blot with a substrate that will luminescent when exposed to the reporter on the secondary antibody.
  • 19. …ANALYSIS Radioactive detection Radioactive labels do not require enzyme substrates, but rather allow the placement of medical X-ray film directly against the western blot which develops as it is exposed to the label and creates dark regions which correspond to the protein bands of interestFluorescent detectionThe fluorescently labeled probe is excited by light and the emission of the excitation is then detected by a photosensor such as CCD camera equipped with appropriate emission filters which captures a digital image of the western blot and allows further data analysis such as molecular weight analysis and a quantitative western blot analysis.
  • 20. APPLICATIONS 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 as above. 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 patients 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. Western blot can also be used as a confirmatory test for Hepatitis B infection. In veterinary medicine, western blot is sometimes used to confirm FIV+ status in cats.