If we add equal concentrations of ConA and glucose to a dilute suspension of cit
ID: 169613 • Letter: I
Question
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 7 (Hemagglutination Analysis of Purified ConA)? 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 with your naked eye.)
*** Lab 7 Background Info (in case needed to answer question): Our experimental variables were ConA + mannose and ConA + galactose. In our protocol we used two 96-well plates (our ConA Dilution plate and the Reaction plate). We made dilutions of our ConA and Control ConA. We added ConA protein to A1 and A2. Then we added ConA buffer to A2 through A12. Our ConA rows were similar to Control ConA rows. In general, we tested the biological activity of our purified ConA protein.
*** Lab 7 Data: Our ConA was expected to have agglutination because we know ConA is able to bind to the sugar residues on red blood cells. In our 96-well plate we saw agglutination present in A1 through A6. However, the ConA concentration becomes so dilute that it's no longer able to agglutinate the red blood cells around A7. Similar results were seen with our ConA + galactose. With ConA + mannose there was interference by mannose so the first couple of wells have agglutination but ConA is eventually not able to agglutinate the red blood cells. The negative control had no agglutination throughout. Ultimately, we were comparing our ConA activity to the manufactured ConA activity.
In summary: Red Blood Cells + ConA ---> agglutination
* ConA = functional
RBCs + ConA:
with galactose ---> agglutination
with mannose ---> partial agglutination
Explanation / Answer
Please find the answers below:
Part 1: If citrate is added with ConA with glucose:
Citrate is an anti-coagulant which prevent coagulation or agglutination of red blood cells. On the other hand, ConA prepared red blood cells for agglutination by binding to their surface sugars and cause them to clump. In a solution carrying equal amounts of ConA and glucose, it is very likely that agglutination will take place even in presence of citrate.This is because when glucose will be present, the total sugar content of the reactio mixture will be high and the red blood cells will agglutinate slowly.
Part 2: If galactose and ConA are added to diluted citrate solution:
In this case, it can be clearly noted that firstly, the anti-coagulant is present at low concentrations so its activity would be slow. Secondarily, ConA is added with a favourable sugar i.e. galactose and thus, according to the reaction as given in information:
RBCs + ConA: with galactose ---> agglutination
agglutination of red cells will be obtained. This need not require a microscope since agglutination reaction can be observed directly by the naked eyes in the form of formation of clump-like thick red structures in the tube.