Question

Western Blotting Homework Assignment (1) (Protected View) - Word intain viruses. Unless you need to edit, it's safer to stay in Protected view Enable Editing Western Blotting Homework Assignment Either write your own one-page reflections of Western Blotting Te answer the following questions. 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 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 electrophores basis does this technique separate proteins? 5. How can you test the efficiency of transfer of the protein from the gel to the nitrocellulose 6. What does the primary antibody recognize? 7. What does the secondary antibody recognize? 8. Which antibody has HRP conjugated to it?

Answer 1

Ansli Klestern blot is a technique which is used to identify specific proteins from a complex mixture of proteins extracted from cells. It require main 3 components in the . Separation by size of molecular weight (in SDS-PAGE). © Transfer to a solid support Identification marking of target protein using a proper primary and secondary antibody to visualise. First, the proteins are separeated by gel electrophoresis. These bands of proteins are then transferred to a membrane. Then this membrane is incubated with &

labels antibodies specific to the target protein. klashing of membrane cause removal of unbound antibody. The bound antibodies are then detected by developing the film. ID0 Applications à . " Can be used to estimate the size. as well as amount of protein pererent in the mixture. It is used as confirmatory test for diagnosis of HIV . It is used for verification of protein production after cloning. Used for analysis of some preotein content » • in serum. . Can also used to Hepatitis & infection confirmatory test for Ans. 2 ce SDS is used to give uniform negative charge to all proteins. Peroteins can be of positive negative or neutral charge. Thus it is necessary to give them same kind of charge for easy separation. . As SDS is negatively charged it binds with protein and denature them to give linearity and uniform charge to that they can be separated solely on the basis of their size.

Ans. 6 Prumany antibody function in Teemany antibody seves different direct and indirect western blot. In direct besteren blot : a perimary for direct detection of because it is conjupated or fluorescent dye. leo antibody is wat target protein with an enzyme 2) In indirect blestern blot - primary antibody is specific to target protein but it not consupated with any - is There Recondary antibody binds to perimany antibody best not to the antigen of target preotein ... e secondary antibodies are labelled with a enzymes or fluorescent dyes to detect bound primary antibody with target protein. Ans.8, Secondary antibody has HRP conperated to it