Practical 3 - measuring protein concentration using ELISA Flashcards
what did the lowry assay measure?
TOTAL protein in our samples
what colour does TMB yield when detecting HRP?
blue
if the unknown protein concentration is read off the x-axis of a standard curve, and we used a dilution factor, what needs to happen next?
need to multiply the x axis value by the dilution factor.
then change units if that value is very large^
an ELISA is an assay used to detect and quantify what?
the concentration of a particular molecule
proteins which act with the analyte (the molecule you are measuring the concentration on) remain…….. to the plate while proteins that are not bound will be……….
remain bound
will be washed away
the antigen is recognised by what??
what then happens to the antigen?
by antibodies labelled with an enzyme
it then becomes immobilized on the plate
in the final/detection stage of the ELISA, what is measured to calculate analyte concentration?
the enzyme catalysed reaction produces a signal which is detected
in the first step of the sandwich ELISA, what binds to the bottom of the well of the 96-plate?
the antibodies which bind free antigen
between each ELISA step, wells are washed with what?
to remove what?
washed with PBS or Tris buffer with Tween detergent
to remove any unbound material/reduce any non-specific binding
how many times should wells be washed between steps to remove any unbound material?
3-5 times
what is the purpose of the blocking buffer with regards to capture antibody and proteins?
to prevent the binding of proteins to areas of the well which are not occupied by capture antibody
blocking buffer reduces background signal and increases assay sensitivity
despite a mixture of proteins being present in the sample, the capture antibody is …….. to the protein of interest, so will only bind to ……….
specific
the protein of interest
The secondary antibody is conjugated to an ………….. so when the substrate for this enzyme is later introduced, a reaction occurs that produces a quantifiable signal. This permits the ……… of the amount of …………… and in turn the amount of …………. in the sample.
enzyme reporter
detection
primary antibody
antigen
Primary antibodies have multiple sites that can be recognized by the secondary antibodies. As a result, multiple secondary antibodies bind to a single primary antibody. what does this do to the signal produced?
amplifies it
the immobilised antigen from the sample is now bound by labelled antibody. What is the next step?
incubate with enzyme substrate to produce a detectable signal
