Hi, we use 2000c Nanodrop to estimate antibody-PE conjugate. We use UV vis method in ND2000c. We see different Abs values at 280nm and 565nm for 1.5ul, 2ul and 3ul. We have an excel which calculate the conc. Of Ab and dye through these Abs values. We see a lot of variations between replicates and between analysts. Suggest if we are doing it wrong way. 1. Which method to use for Ab-dye sample. 2. Why there is a difference in Abs values with 1.5ul, 2ul and 3ul. 3. Why Abs values are not repetitive from a single sample. Thanks
Nano is very sensitive to cleanliness,volume ,zeroing/blanking and pH
Ensure that the pedestal has been cleaned of absorbed protein,dna and detergents by wiping with HCl and washing with water.Check that 1ul of water on the pedestal does not spread across the pedestal but forks a bead or droplet on the pedestal.Spreading indicates protein/detergent contamination of the window. Make sure that the blanking solution is exactly the solvent that the samples are dissolved in. Make sure that the samples are not so dilute that they fall below the minimum accurate measurement limit of 0.4ng/ul.
Run a spectrum to make sure that you are measuring in a flattish and not a steep part of the spectrum as +/- 1nM in wavelength in the steep part of the spectrum makes a big difference.
Test your users .These are small volumes and poor pipettes/pipetting makes a big difference. get them to quickly pipette 20 volumes of 1ul water into a tube on a balance and see that it weighs 20ug.
Check that your users are measuring at the same delay time after loading the sample...leaving a sample on too long may absorb CO2 from the air,change the pH and give different readings for different users or between replicates that have different times on the pedestal
Thank you @Paul Rutland for your descriptive responses. Will surely consider your suggestions and hope an improvements.
Arun, I would take a high quality sample to test its reliability. Assuming the nanodrop reliability checks out, I would consider that your sample may be non-homogeneous. The most likely problem would be microparticles (i.e. dust, powder from micro-columns, etc...). Try centrifuging your sample before taking out an aliquot for measurement to pellet any particulates.
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