Tables SmartArt Revie Book Tele lust Paragrmph 1. (15pts) In the cell bio lab, w
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Tables SmartArt Revie Book Tele lust Paragrmph 1. (15pts) In the cell bio lab, we use company manufactund gels, however you can make you own polyacrylamide gels. List all of the ingredients found in an SDS-PAGE gel. Which ingredients are responsible for polymerizing the solution? How does the percentage of acrylamide effect the migrati 2. (10pts) If we add equal concentrations of ConA and glucose to a dilute suspension of citrated red blood cells in ConA buffer, what should we expect to happen based on our data from Lab ?Explain why we expect to see these results. Also, if we added equal amounts of ConA and galactose to a dilute suspension of citrated red blood cells in ConA buffer, what should we expect to happen? Explain your answer. (Describe what is happening at the cellular and or molecular level rather than what you actually see naked eye.) 3. 20pts) Describe two common uses for bemagglutination assays are in healthcare and research. What induces agglutination in each scenario? Why does the agglutination occur in cach scenario? 4. (45pts) You purified protein x via affinity chromatography (no diafiltratinn step performed) and ran an SDS. PAGE gel of the sample with a set of controls. Below is the result of your sDs-PAGE analysis. 170 Figure 1.SDS-PAGE of purified protein x Lance 1. Protein ladder (in Daltons Lane 2, purified protein x (affinity tography) Lane 3, purified protein x(company manufactured). Lane 4, elution buffer a. What is the benefit of a protein ladder molecular weight marker in an SDs AGE gel? Describe what you can eam about the protein bands found in lanes 2.3 and 4 based on the protein ladder bands,Explanation / Answer
1. (i) SDS-PAGE electrophoresis is widely used in separating proteins of a sample. Though there are many components used for performing an SDS-PAGE, here we will discuss the ingredients required for preparing the gel.
It is important to note, that SDS-PAGE consist of a running gel and a stacking gel, both have a different purpose.
Ingredients required: Acrylamide (polymer), Bis-acrylamide (cross-linker), SDS (anionic detergent), Ammonium persulphate or APS (free radicals), TEMED (stabilizer), Tris-HCl buffer at pH 8.8 and pH 6.8 and lastly, water.
(ii) Acrylamide is the polymerizing compound, which with the help of bis-acrylamide is able to cross-link and form gel. Greater the percentage of the acrylamide, smaller and finer would be the pores of the gel. Hence proteins will easily migrate in a gel with 4% acrylamide and migrate slowly in a gel with 18% acrylamide.
2. Con A is known to agglutinate red blood cells. ConA does so, by binding its receptors to the glucose residues on the membrane of red blood cells.
Citrate or citric acid is an anti-coagulating agent and citrated red blood cells in a dilute solution are thus protected from coagulation.
(i) So, from the above information, we can predict that when equal concentration of ConA and glucose are added to a dilute solution of citrated red blood cells in ConA buffer, there will not be any agglutination. The free glucose molecules will compete with the membrane-bound glucose molecules for binding with ConA.
(ii) Galactose does not interfere with the agglutination of ConA with red blood cells. Thus, if the purified ConA is active,, in the presence of galactose, there will be partial agglutination between ConA and red blood cells. The agglutination would be partial because of the presence of citric acid.
3. Hemagglutination is the process of agglutination of red blood cells. It is used for the purpose of (i)Blood typing and (ii) Viral hemagglutination assays
(i) In blood typing, anti-A, anti-B, and anti-Rh antibodies are taken in three wells. The blood sample of an individual is mixed in each of the three wells and observed for agglutination. In this way, it is determined whether the person's blood group is A, B, AB or O.
(ii) Some viruses, such as the influenza virus, infects by binding to red blood cells and causing agglutination. This can be determined by hemagglutination assays.
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