LIGAND-RECEPTOR BINDING - RADIOLABELLED LIGAND

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tazuk
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LIGAND-RECEPTOR BINDING - RADIOLABELLED LIGAND

HI
 OK I HAVE A LIST OF RESULTS OF 24 TUBES AS DPM (DISINTEGRATIONS PER MIN)
 
ALL TUBES HAVE RADIOLABELLED LIGAND CONC IN PMOLES. AND MEMBRANE SUSPENSION AS 1.0MG.
 TUBES 13-24 HAVE THE UNLABELLED 'COLD LIGAND IN MICROMOLAR.
I NEED TO WORK OUT TOTAL, NON-SPECIFIC AND SPECIFIC BINDING IN PMOLES/MG AT THE 4 CONCENTRATIONS USED ~ 1, 4, 8, 15PMOLES.
SPECIFIC ACTIVITY IS 1.157 x 10^5 OF LIGAND IS [3H]NALOXONE (SHOULDNT MATTER)
 AFTER CALCULATION TOTAL/ NSB I NEED TO DISPLAY IT GRAPHICALLY... HOW DO I DO THIS, WHAT ARE THE AXIS >?
HELP PLS

R Bishop
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Hi tazuk,

Hi tazuk,
Yeah thats a tough one.  Here's the way I see it based off the info you gave us.  
The best way to see the increase in binding is to graph the x -axis as increasing concentration of pmole [3H]NALOXONE (1,4,8,15).  The y-axis will end up being pmole NALOXONE/mg membrane protein or essentially pmoles bound since you are adding 1mg of membrane protein.  Of course you derive pmoles bound from your radiospecific activity.
 
Non-specific is the pmoles bound in the 1000-fold excess cold samples. You then subtract the non-specific from the total to achieve specific binding.
 
Hope that was clear.  I use Prism 4 by GraphPad to graph these types of assays typically, but any graphing software could do it.
 
Rus

tazuk
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 Thanks

 Thanks
but how do i convert my data..dpm into data for my y-axis (pmole/mg) 
using what i have the specific activity as stated  and the 1.0mg of membrane suspension
do i take average from readings as there are 3 tubes for each conc.
or is my total binding curve just simply conc on x axis and dpm on y axis?
do i have to graph it out to calculate bindings...so i need to convert dpm  using SA= 1.157X10^6 dpm/pmol  into>>>>>>>>>pmoles/mg 
 
.after calculation total, non-specific i have to graphically show it using Excel...then make a scatchard plot then caluclate Kd Ro and so on..
 appreciate the help

R Bishop
R Bishop's picture
 Sorry for the delay, busy

 Sorry for the delay, busy morning today. 
 
I think you have it right. Counts recovered/specific acitvity should give you pmoles recovered. You have 1mg extract so that should give you pmoles/mg.  I would input all three data points into the graphing software and present the mean +/- standard deviation.  just take the mean of the non-specific.
 
Once you get your graph then you can calculate Kd etc.  I think excel might even do it for you.  I use Prism by Graph pad and I know that will do it automatically.
 
Rus

tazuk
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Hi Rus,

Hi Rus,
thanks very much for your help
I've attached an excel file if u have a min to check what ive done.
Still having some trouble graphing the data out. and still working at it
I think i've done the calculations ok.
After this graph the next agenda will be constructing a scatchard plot and calculating Kd and R0 Then producing a Linweaver burke plot and comparing them.
 

R Bishop
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I cant open the rar file?

I cant open the rar file?
Rus
 

The FFM
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rar is like a .zip file but

rar is like a .zip file but it uses the WinRAR program to compress the file.
 
Anyway tazuk, unless it is being specifically demanded of you you dont need to do a Lineweaver plot these days as they are prone to error, although they do offer a nice way to easily visualize the Km and Bmax etc.
You can use your graphing software to do a 1 site bind Michealis-Menten non-linear regression analysis to fit your data.  Plots the Mean +/- standard error of the specific activity for each concentration on the Y-axis vs the [Naloxone] on the x-axis. Use the fit to obtain the Km and Bmax.
 
If you still want to do it the old fashioned way to display the data in an easy to visualize form, here is a nice description of how to do a Scatchard Plot (http://www.curvefit.com/scatchard_plots.htm)
and on the same site, here is the reasons why to avoid doing the transformations when performing the actual calculations www.curvefit.com/avoid_linearizing.htm