Representative image of a processed array
Bar graph quantifying signal intensities
Bar graph quantifying percent CVs
Equipment: Nano-Plotter NP2.0
Courtesy of HTS Resources , San Diego (USA)
Common Recommendations for Spotting of Antibodies:
- Use protein concentrations of max. 1 mg/ml containing less than 1 M salt.
- As proteins need higher piezo voltage, activate the stroboscope break in the standard NPL programs to adjust the parameters. Do not print solutions requiring varying conditions, this is difficult to handle and results in spots of different size.
- Carbohydrates like trehalose can help hydrate proteins and maintain their native structure even in a dry state. But you must prove that these viscous solutions can be spotted without problems.
- If you have a large protein supply, ultrafiltration would remove aggregates. If you have only small volumes, centrifuge at least.
- Avoid to suck particles into the pipettes by not using up the entire sample volume.
- Added buffer should be sterile-filtered to prevent spoiling.
- The first spot in a row of spots may be stronger than the following ones. If this is the case, define a “yellow paper object” near your first slide and dispense the first spots onto this target.
- Adjust the piezo parameters for each pipette before each run until the droplet pattern in the stroboscope “looks good” for all of them. Your experience is needed here.
- If you require low inter-tip CVs or need to know absolute droplet volumes, dispense labeled protein and quantitate spots in a scanner.
Source: Manual for NP2 Systems, Edition 2007