Quantitate Sample Video
Final Voice-Over Script
1/16/13
In this video, we will show a procedure for fast quantitation of a protein of
interest using the innovative BLItz system from Pall ForteBio.
The BLItz system is an easy-to-use personal benchtop assay system for label-free
detection and analysis of proteins. BlItz is based on the same biolayer
interferometry technology as ForteBio's Octet platform, and works with just
microliter amounts of sample. BLItz can perform rapid detection, quantitation,
kinetics, and assay development in a simple Drop-Read-Done format.
INTERVIEW: Introduction
This experiment will demonstrate how to quantitate antibodies expressed in cell
culture supernatant, using a previously saved standard curve.
In order to perform a quantitation experiment, you must first generate a
standard curve with your protein or antibody of interest using the Create
Standard Curve module in BLItz Pro software. Refer to the BLItz training video on
Creating a Standard Curve for more details.
You will need a BLItz system connected to a laptop computer running BLItz Pro
software, Protein G biosensors, cell culture media, and the unknown samples to
be quantitated. Also required are microcentrifuge tubes, a 96-well flat-bottom
microplate, pipettors, pipet tips and lint-free laboratory wipes.
Be sure to equilibrate all reagents and samples to room temperature prior to
use.
Turn on the BLItz instrument and allow to warm up for at least 30 minutes.
Launch BLItz Pro software and click on the Quantitate Sample icon. You are now
ready to begin the procedure.
The disposable Protein G Dip-and-Read biosensors come ready to use with
immobilized Protein G. The assay is performed on the tip of the biosensor, so it
is important to avoid touching the tip to any solid surface.
Biosensors must be hydrated prior to use in a solution that matches the matrix
of your samples. For quantitating antibodies in cell culture supernatant, hydrate
biosensors with the same cell culture media.
Three Protein G biosensors will be required to quantitate two unknown samples.
One will be used for a negative control.
Pipet 200 microliters of culture media into wells of the 96-well microplate that
correspond to the biosensors to be used. Remove the lid of the biosensor tray,
then carefully lift the biosensor rack out of the holder. Place the 96-well
microplate into the holder and replace the biosensor rack to immerse the tips of
the biosensors in the liquid.
In the software window, select a hydration time of 10 minutes. Click the Hydrate
Biosensor icon in the software window to start the hydration timer.
Enter the run settings in BLItz Pro software.
Enter an experiment name and description
Enter sample ID and concentration. Start with the negative control sample first.
The negative control will be used to subtract background signal and must be
identical to the matrix of your samples. For this experiment we will use cell
culture media without antibody.
Select Protein G under biosensor type.
Enter a run time of 30 seconds. A longer run time may be used, but should not
exceed 300 seconds. The run time must also match the run time of the Standard
Curve to be used.
INTERVIEW: Considerations for successful experiment
Click the Next icon in the software window. The Run Data chart, Run List table
and Standard Curve chart will appear. A series of prompts will pop up to guide
you through the assay.
To load a sample, first open the instrument cover.
Carefully pipet 4 microliters of cell culture media into the drop holder. Be sure
not to expel air from the pipettor as this may introduce bubbles.
Remove a hydrated biosensor from the tray and push it firmly onto the
biosensor mount. Once in place, give the biosensor a quarter-turn while
pushing upward to make sure it is seated well.
Check that the slider is in the right-most position so that the biosensor is aligned
to the drop holder.
Close the instrument cover to start data acquisition automatically.
Data is collected in real time on the sensorgram visible on-screen. Once
acquisition is complete, a tone will sound.
When the Save As dialog box appears, enter a file name and save the
experiment file.
Open the instrument cover and discard the used biosensor.
Wipe the drop holder dry with a laboratory wipe. Rinse 3 times with water or
buffer, wiping dry each time with a laboratory wipe.
Repeat the procedures used for the negative control to run the first unknown
sample.
Enter the Sample ID and click 'Next'.
Pipet 4 microliters of sample into the drop holder. Mount a new hydrated
biosensor, and close the cover to automatically start data acquisition.
Observe that the unknown sample shows a positive response compared to the
negative control, indicating the presence of antibody.
Once data is acquired, rinse the drop holder as before.
Repeat the same procedures for the remaining sample, using a new hydrated
biosensor.
When data acquisition is complete for all samples, wipe the drop holder dry and
rinse with 4 microliters of point 5 normal hydrochloric acid solution, followed by
2 rinses with water. This will deep clean the drop holder and prevent carryover
into the next experiment.
Check the Run List table to see binding rates for each of the samples A higher
binding rate corresponds to higher concentration of target protein. Check the
Reference box next to the negative control sample to automatically subtract
background signal.
To load a previously saved standard curve, click the Load Standards button
above the Standard Curve chart. Select the standard curve file that corresponds
to your samples.
The software automatically plots the binding rates for the unknown samples
against the standard curve and calculates concentration values. On the chart,
standards are plotted in blue, unknown samples in red. The calculated
concentration values can be viewed in the Run List Table.
Click Create Report to generate a pdf document recording the results.
INTERVIEW: Summary/conclusion