Question

100 1. How western blot does works and explain some of its possible application? 2. While using the sample buffer (Leammli buffer), there was SDS (detergent). Why was the SDS necessary for running the gel? 3. Why was it necessary for the transfer to the nitrocellulose membrane? 4. The first stage of western blotting involves separating proteins using gel electrophoresis. On what basis does this technique separate proteins? 5. How can you test the efficiency of transfer of the protein from the gel to the nitrocellulose membrane? 6. What does the primary antibody recognize? 7. What does the secondary antibody recognize? 8. Which antibody has HRP conjugated to it?

Answer 1

1)The western blot is a biological analytic technique also known as protein immunoblot as it is used in protein detection.
In western blotting immunochromatographic principle is used in which biological protein is passed through polyacramide gel matrix and collected on nitrocellulose membrane using blotting apparatus. This separated proteins are then detected using primary antibody and secondary enzyme reaction.
It involves 3 steps:

A ) Separation of Proteins: Electrophoresis through gel matrix is used for separation of protein. Polyacramide gel is used for gel matrix. Protein mixture is runned through this gel matrix, which gets separated on to the membrane depending upon the size (length of linear chain), molecular weight and charge.
To gel matrix SSD is adden to uncoil the protein structure and made it linear chain.

B) Blotting: It is a process in which separated proteins are collected on solid support. In this method Nitrocellulose membrane or nylon membrane is used. It is necessary to collect proteins on nitrocellulose membrane for antibody staining and protein detection based on hydrophobic interaction.

C) Antibody staining and protein detection: On membrane proteins gets separated and forms protein bands. This protein bands are blocked by using primary antibodies which are specific to our target protein. Unbound antibodies are washed off and now only anti bodies bounded to our target proteins are left on the membrane. Now this membrane is treated with secondary enzyme which makes bands visible. thickness of band is directly proportional to amount of protein.
visible band are now detected using various techniques.
In autoradiography procedure is used where protein bands are bounded to radioactive antibodies and visible bands are seen by exposing membrane to X-ray film. ( Radioactive detection)
usually enzyme linked antibodies against protein is used. ( Calorimetric detection)

Applications
* It is used is HIV detection test where it helps in detection of anti hiv antibody in human serum
* To detect the size and amount of protein in mixture.
* To detect the specific protein in a complex mixture of proteins.
* Detection of specific anitbodies in case of neurocysticercosis and also in tubercular meningitis.

2) Electrophoresis through gel matrix is used for separation of proteins or DNA. DNA Structure is helical or some proteins also exist in coils form specially in case of tertiary structure. Its complex structure is bonded by disulphide bond, it also contributes to some charge on molecule. Smaller proteins and simple structures can easily migrate through gel matrix but also show some resistance due to charge and in case of complex protein its coiled structure and charge causes slow movement of proteins through gel matrix.
Hence, for ease of separation some denaturating agents are added which breaks the disulphide bonds and make structure of protein easy to flow.
In Gel Electrophoresis while using leammli buffer SDS - Sodium dodecyl sulphate which is also known as sodium lauryl sulphate is added which has denaturing effecct . This detergent bind to backbone of protein and along with reducing agent cleaves disulphide bond which unfolds proteins into linear chain .

3) It is must to transfer separated proteins on nitrocellulose membrane as we need to probe or stain it using primary antibody which forms visual protein bands then using secondary enzyme detection is done.

4) In gel electrophoresis proteins get separated based on molcular weight.Its separation os also effected by size, structure and charge.

6) Primary antibody are specific to our target proteins which block them on membarne and other proteins gets removed. it forms bands of proteins .
7) Secondary antiodies makes that proteins band which are bounded to primary antibodies visible.