Western blotting


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Western blotting

  2. 2. CONTENTS Tissue preparation Gel electrophoresis Transfer Blocking Detection Analysis Applications
  3. 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. 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. 5. GEL ELECTROPHORESIS The proteins of the sample are separated using gel electrophoresis We use SDS-PAGE(sodiumdodecyl sulphate-polyacrylamide gelelectrophoresis).
  6. 6. *Sampledproteins becomecovered in thenegativelycharged SDSand move to thepositivelychargedelectrodethroughthe acrylamidemesh of the gel.
  7. 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. 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. 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. 10. ..PAGE Small molecules move through the polyacrylamide forest faster than big molecules. Big molecules stays near the well.
  11. 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. 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. 13. Protein gel (SDS-PAGE) that has been stainedwith Coomassie Blue.
  14. 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. 15.  when the antibodyis added, there is noroom on the membranefor it to attach otherthan on the bindingsites of the specifictarget protein.
  16. 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. 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. 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. 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. 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.