Antibody Arrays without Cross Contamination

Antibody Pipetting with Piezoelectric GeSiM Tips

  • 4 antibodies were assessed (IL6, EGF, PSA, C-RP)

    Rows of protein spots before and after tip washing

    Rows of protein spots before and after tip washing

  • Antibodies were spotted at 100ug/mL concentration
  • To evaluate carry over between each antibody two plain water samples were aspirated (Water Wash 1 and 2)
  • The GeSiM Nano-Tip was used at one drop (approx 350 pL) per spot
  • Arrays were spotted on GenTel PATHTM slides
  • The tip was washed with water (9 seconds), 0.2N KOH (2 seconds) then water (9 seconds). The piezo was activated during the  water washes
Bar graph quantifying signal intensities

Bar graph quantifying signal intensities

Bar graph quantifying percent CVs

Bar graph quantifying percent CVs


Courtesy of HTS Ressources, 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. In case of varying spotting parameters for different samples optimized values should be added to the well plate file (See manual for more instructions).
  • 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.