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You are assigned a set of data which represents readings from the following experiment to determine the affinity of an antibody for an antigen (hapten):A measured amount of the antibody was placed in side 1 of a number of dialysis chambers and additions of radioactive antigen were added to side 2 of the chambers and the systems allowed to come to equilibrium.  Samples for counting were then taken from both sides of the membranes and counts obtained in a scintillation counter.  Specific activity of the radioactive small antigen was 1015 cpm/mole (counts per minute per mole).  Data obtained are cpm/ml.Data Set 50Side 1Side 239443504388060477137051060805400790566211007242220079891300842704008716650089236600907997009202880093025900938571000Aim: Present a plot of Bound v. free antigen showing the saturation curve of your data. Then derive and plot the data as a Scatchard plot and estimate the affinity of the antibody for the antigen.  Express the affinity as an affinity constant (Ka) with appropriate unit.  Present your analysis as for an experimental report showing in full how you used your data to obtain your affinity constant. You must show the direct plot and sample calculation as well as the Scatchard transformation that you used to estimate the Ka.Presentation: Title “Scatchard Analysis”.  Student name top right corner with data set number underneath.  NO DROP BOX please.  Hard copy required unless permission granted for email submission.Deadline:  Friday 28 Sept., 5 pm.  Late penalty 10% of marks per semester day.Scatchard AssignmentQ: I have been through the lecture notes to do the Scatchard assignment and I have a question: On one of the slides that shows a beaker with side one and side two, there are a number of equations relating to the various concentrations of components. One such component is [Ab]t yet this is the only reference to it and I would not be able to figure out what it is based upon the other equations/information given (unless I’m missing something). Does this quantity actually refer to the Ab term in [Ab.Ag] as if so then I can figure it out.A: With the “single point” equilibrium dialysis experiment you must obtain a value for free antibody as this is one of the terms in the equilibrium equation for Ka.  This can only be done if you know the concentration of total antibody that has been put into the compartment.  Knowing total antibody allows calculation of free antibody by subtracting the concentration of complex [Ab.Ag] from the total antibody.  So no, this quantity is not the Ab in the [Ab.Ag] term.  The total Ab is [Ab.Ag] + Ab(free).The point of the Scatchard analysis in the assignment is that you do not have to know the total antibody to do this analysis.  By varying the free antigen the dependence of [Ab.Ag] on [Ag] is found and this gives Ka.Q: When I prepared the saturation curve of my data, I noticed that the point of saturation had not yet been reached (i.e. it was still in the linear range of the curve).  I don’t think I have made any errors, and have put other data sets into my spread sheet, and noticed that they all reach a saturation point.  How should I go about calculating Ka from this plot now? I could extrapolate, then take the inverse of the maximum point etc, though this would be particularly inaccurate (even more so than normal)..I was wondering whether my data set actually had an error in it, considering that my data should theoretically be ‘ideal’?A: The “point of saturation” is never reached.  It can only be reached by extrapolation to infinite antigen concentration.  What you mean is that your data is lower on the saturation curve than the other data sets you have looked at.  You do not need to extrapolate the data or find the “saturation point”.  The Ka is obtained by using the Scatchard transform, plotting the data and obtaining the slope of the line, the value of which is the Ka.  No, your data does not have an error in it  

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