Cell Biology Lab 7
PCB 4023 L
TA: Sushmita Mustafi
Quantifying the assay.
Cells absorbing sarrounding/
Extracellular particles, small
Degraded food particles,
the absorbed particles are non
Small vesicles helps in
Pinocytosis which finally
Fuses with lysosomes as well.
Fluid phase marker: HRP
Fluid phase endocytosis : Pinocytosis
Marker: To track the fluid that has been
HRP or Horse Radish Peroxidase is used as
a fluid phase marker.
Enzyme that catalyze the oxidation of a
ROOR' + electron donor (2 e-) + 2H+ →
ROH + R'OH
From plant Horseradish.
Needs Hydrogen peroxide [H2O2] as oxidizing
Commonly used in ELISA/
CONJUGATES for its characteristic color
change reactions with specific substrates.
O-phenylenediamine ----------- 2,3 Diaminophenazine.
[ Color less] [Orange brown]
Measure amount of HRP from the color
--------- transmitted light
Absorbed light is proportional to
concentration of solutes the solution.
Measuring in a spectrophotometer:
Higher concentration of solutes -> higher absorbance, lower
Blank : contains all other additional reagents that are not
responsible for the actual color change.
Ex. In an enzyme/ substrate reaction: higher conc of enzyme =
Higher conc of product.
Hence measuring the product conc is indirect way of estimating
the enzyme conc.
But what about all other reagents that help in the enzyme
reaction? They have some absorbance that could add up with
the absorbance of the product.
Hence to cancell the absorbance by secondary reagent, we
Eclone cells mediated Pinocytosis at time
0min/ 5min/ 10min/ 20min/ [ 37c incubator]
Use 0.2% tritonX to lyse the cells and collect
Part B: Enzyme assay
OPD dissolved in NaHCO3 and H2O2.
Add OPD to cell lysate and let them react at
room temperature. Observe the color change.
Stop the reaction with 0.1N Sulfuric acid.
Read the absorbance.
Observing the rate of Pinocytosis at different