Practical 5-cAMP Binding Assay

what are ligand binding assays for?

measure interactions between 2 molecules

advantages vs disadvantages of fluorescence based assays

broad spectrum of wavelengths and multicolouredd

• issues with interference, ligand alters after binding

what is a radioligand?

radioactively labelled molecule that binds specifically to a target protein

what is a saturation binding assay?

measures equilibrium binding- increasing radioligand concentration to a fixed amount of receptors to measure Kd and Max

what is a competitive binding assay?

measures the ability of an unlabelled ligand to compete with the radioligand for binding to determine Ki

what is a kinetic assay?

measure Don and Koff- rates of which the ligand binds and dissciated with receptors

what is Bmax and kD?

• bmax- number of receptor binding sites

• kd- dissciation cnstant- low Kd- high affinity- Kd is ½ of the Bmax

describe a scatchard plot- what does it measure etc

• x axis- ligand bound

• y axis- bound:free ratio

• slope= -1/Kd

• x intercept= Bmax

what is specific vs non specific binding?

specific- radioligand binding to the receptor of interest

non specific- radioligand binding to other proteins etc

how is non specific binding measured?

add extra competitor to displace the radioligand from specific sites

how is radioligand measured and difference between CPM and DPM and how is it calculated?

• by LSC- liquid scintillation counting

• DPM= disintegrations per min, CPM=counts per minute

• COM/DPM x 100%

what is quenching?

loss of energy transfer from the radioactive decay to the light

how does adding non radioactive cAMP affect the assay?

increases the total cAMP, dilutes radioactivity and lower CPM

how is percent control binding calculated?

• CPM of the sample/mean CPM of tubes with no cold cAMP x 100

difference between cold cAMP and Hot cAMP

cold- non radioactive and normal cAMP- competes with hot cAMP

hot- radioactive and measured

what does theophylline do in the buffer?

stops the breakdown of cAMP to 5’AMP

what does charcoal do?

bind to free cAMP from the bound cAMP

how would you calculate the concentration of radio labelled cAMP of vials 19 and 20

find the mean of 19-20

actual dpm= cpm/counter efficiency

dpm to curies= dpm/2.22×10^12

curies to moles= curies/Ci/mmol value

molarity= moles/volume in L

if 50microL of stock and final vol in 300microL= 1 in 6 dilution, molarity/dilution factor