Cell Bio Lab Report
TITLE AND AUTHOR
Lab 7 Analysis of purified Concanavalin A via:Hemagglutination INTRODUCTION The purpose of this lab was to test the biological activity of ConA by performing a hemagglutination assay. If ConA is active then agglutination will occur due to ConA’s free receptors being able to bind to the glucose residues on the sheep’s red blood cells. If ConA is not active then no agglutination will occur. To test the hemagglutination reaction, two types of ConA solutions were compared, a purchased control ConA solution in buffer as the positive control, and a purified solution of ConA in buffer previously purified in lab. Each solution was at a 2mg/ml concentration of ConA in ConA buffer, which is necessary for ConA's biological activity. Two variables were added, Galactose and Mannose, to the ConA solution to compare the effects each had on the hemagglutination reaction. I hypothesize for ConA to be able to agglutinate the red blood cells if in the adequate concentration and if in the presence of Galactose, not Mannose. Mannose will inhibit the ConA from binding to the red blood cell’s membrane, preventing agglutination.
RESULTS
The reaction plate containing the ConA dilutions was incubated over the weekend and resulted in all wells being pink, appearing as if every well had agglutinated. There was a vague outline of the non-agglutinated cells in various wells. The last agglutination was observed at titer 0.0625 (1/16). Agglutination was seen in rows A, B D, and E (row A contained the control ConA, row B contained the control ConA + Galactose, row D contained the sample ConA, and row E contained the sample ConA + Galactose). In the well rows C and F which contained control ConA + Mannose and sample ConA + Mannose, agglutination did not occur at any concentration of ConA. Row G, the negative control appeared to have agglutinated as well as Row H, which contained only ConA buffer. DISCUSSION AND CONCLUSION The results
Lab 7 Analysis of purified Concanavalin A via:Hemagglutination INTRODUCTION The purpose of this lab was to test the biological activity of ConA by performing a hemagglutination assay. If ConA is active then agglutination will occur due to ConA’s free receptors being able to bind to the glucose residues on the sheep’s red blood cells. If ConA is not active then no agglutination will occur. To test the hemagglutination reaction, two types of ConA solutions were compared, a purchased control ConA solution in buffer as the positive control, and a purified solution of ConA in buffer previously purified in lab. Each solution was at a 2mg/ml concentration of ConA in ConA buffer, which is necessary for ConA's biological activity. Two variables were added, Galactose and Mannose, to the ConA solution to compare the effects each had on the hemagglutination reaction. I hypothesize for ConA to be able to agglutinate the red blood cells if in the adequate concentration and if in the presence of Galactose, not Mannose. Mannose will inhibit the ConA from binding to the red blood cell’s membrane, preventing agglutination.
RESULTS
The reaction plate containing the ConA dilutions was incubated over the weekend and resulted in all wells being pink, appearing as if every well had agglutinated. There was a vague outline of the non-agglutinated cells in various wells. The last agglutination was observed at titer 0.0625 (1/16). Agglutination was seen in rows A, B D, and E (row A contained the control ConA, row B contained the control ConA + Galactose, row D contained the sample ConA, and row E contained the sample ConA + Galactose). In the well rows C and F which contained control ConA + Mannose and sample ConA + Mannose, agglutination did not occur at any concentration of ConA. Row G, the negative control appeared to have agglutinated as well as Row H, which contained only ConA buffer. DISCUSSION AND CONCLUSION The results
Cited: Cell Biology 3822 Lab Manual, Cell Surface Glycoprotein Receptor Analysis Using Concanavalin A Lab 7. Pearson Learning Solutions. 2012: 147-154. Madeleine Zaechringer. Cell Biology 3822 Analysis of purified ConA via Hemagglutinatino Assay Lab 7: Powerpoint. 2014.